Denitrifying and dephenolizing bacteria and its application in treatment of sewage containing nitrogen and phenol

Abstract

The application belongs to the technical field of wastewater treatment, and particularly relates to a denitrifying and phenol-removing bacterium and application of the bacterium in treatment of wastewater containing nitrogen and phenol. Acinetobacter gaillouiae The bacterium is strain BY18, and the preservation number is CCTCC NO: M 20232439. The strain BY18 of the application can simultaneously and efficiently remove phenol and inorganic nitrogen, and the maximum removal efficiencies are as follows: ammonium nitrogen and phenol: 4.36 mg / L / h and 62.20 mg / L / h; nitrate and phenol: 2.91 mg / L / h and 68.22 mg / L / h; nitrite and phenol: 4.72 mg / L / h and 60.82 mg / L / h.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a denitrifying and phenol-removing bacterium and its application in the treatment of nitrogen- and phenol-containing wastewater. Background Technology

[0002] In recent years, the excessive accumulation of nitrogen compounds has caused enormous harm to aquatic ecosystems and human health. For example, excessive ammonium nitrogen in water bodies can lead to eutrophication, blackening and foul odor of the water, and a decrease in dissolved oxygen levels, resulting in poisoning and even death of aquatic organisms. Long-term consumption of water contaminated with nitrates or nitrites may lead to methemoglobinemia and certain cancers such as stomach, esophageal, and liver cancer. On the other hand, industrial and domestic wastewater from coal mining, chemical pesticides, leather processing, printing and dyeing, and coking not only contains high concentrations of inorganic nitrogen compounds but also complex and persistent organic matter. Phenol, in particular, is a highly toxic and persistent organic compound that has a strong corrosive effect on the skin and mucous membranes, easily causing tissue necrosis and damage. It has been included in the "blacklist" of priority pollutants for treatment in China and many European countries. The toxicity level of phenol to humans is usually between 10-24 mg / L, and to fish between 9-25 mg / L. However, the concentration of phenol in various industrial wastewaters varies from 2.8 to 6800 mg / L. In addition, regulations stipulate that the concentration of ammonium in drinking water should be less than 0.5 mg / L, but in some polluted water bodies, the ammonium content exceeds 2 mg / L.

[0003] Some physicochemical methods, such as electrochemical oxidation, organic solvent extraction, and activated carbon adsorption, have been reported for treating wastewater contaminated with inorganic nitrogen and phenol, but these methods are more expensive and prone to secondary pollution compared to biological methods. Early methods commonly used anaerobic ammonia oxidation and anaerobic denitrification to purify nitrogen and phenol-contaminated wastewater, with phenol and inorganic nitrogen acting as electron donors and acceptors, respectively, to achieve simultaneous removal of phenol and inorganic nitrogen. For example, Magnetospirillum spp. CC-26 can utilize phenol as a single carbon source for anaerobic denitrification. However, phenol stimulates bacteria to activate self-protective mechanisms, inhibiting the activity of anaerobic denitrifying enzymes and anaerobic ammonia oxidizing enzymes, affecting the microbial community structure and biomass within the reactor. Compared to anaerobic biodegradation, aerobic biodegradation is more conducive to the complete mineralization and removal of phenol. Furthermore, a large number of heterotrophic nitrifying-aerobic denitrifying (HN-AD) bacteria have been reported for treating nitrogen-containing wastewater. For example, *Pseudomonastaiwanensis* EN-F2 achieved removal rates of 100%, 89.21%, and 86.99% for ammonium nitrogen, nitrate nitrogen, and nitrite nitrogen at an initial concentration of 50 mg / L, respectively. *Streptomyces mediolani* EM-B2 achieved maximum removal rates of 3.46 mg / L / h for ammonium, 1.72 mg / L / h for nitrite, and 1.71 mg / L / h for nitrate. However, studies on these denitrifying bacteria primarily utilize simple organic sources such as glucose, sodium acetate, and sodium citrate. Although persistent organic pollutants (such as phenol) and nitrogen compounds coexist in various wastewaters, few studies have focused on the removal of these persistent organic pollutants. In fact, a few studies have reported the degradation of phenol pollutants in wastewater, but the denitrification performance of the strains has not been investigated, and most studies have concentrated on utilizing mixed bacterial communities in activated sludge reactors and biofilm reactors. Some studies have reported the use of single strains for nitrification and denitrification using phenol as a single carbon source, but the removal rates of both phenol and inorganic nitrogen are very low. For example, the simultaneous degradation rates of phenol and nitrate by Pseudomonas sp. HJ3 were only 3.76 mg / L / h and 0.76 mg / L / h, respectively. Similarly, Serratiasp. LJ-1 removed 100 mg / L of ammonium and 400 mg / L of phenol at removal rates of only 0.67 mg / L / h and 1.6 mg / L / h, respectively, over 150 h and 250 h. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a denitrifying and phenol-removing bacterium and its application in the treatment of nitrogen- and phenol-containing wastewater.

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

[0006] In the first aspect, the present invention provides a strain, namely AcinetobactergaillouiaeBY18, with accession number CCTCC NO: M 20232439, which was deposited at the China Center for Type Culture Collection on December 4, 2023, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] Secondly, the present invention provides the application of the strains described above in the treatment of phenol-containing and / or nitrogen-containing wastewater.

[0008] Thirdly, the present invention provides the use of the strains described above in the preparation of products for treating wastewater containing phenol and / or nitrogen.

[0009] In a fourth aspect, the present invention provides a water treatment agent comprising the strains described above.

[0010] Fifthly, the present invention provides a wastewater treatment method, the wastewater treatment method comprising: inoculating the strains described above into the wastewater to be treated and culturing them.

[0011] Optionally, the inoculum size of the strain is 0.2*10⁻⁶. 8 CFU / mL -0.7*10 8 CFU / mL.

[0012] Optionally, the culture temperature is 20-30℃ and the culture pH is 6.2-8.2.

[0013] The beneficial effects of this invention are as follows:

[0014] The strain BY18 of this invention can simultaneously and efficiently remove phenol and inorganic nitrogen, with maximum removal efficiencies of: ammonium nitrogen and phenol: 4.36 mg / L / h and 62.20 mg / L / h, respectively; nitrate and phenol: 2.91 mg / L / h and 68.22 mg / L / h, respectively; nitrite and phenol: 4.72 mg / L / h and 60.82 mg / L / h, respectively.

[0015] The strain BY18 of this invention has broad-spectrum environmental adaptability.

[0016] In this invention, compared with nitrates and nitrites, using ammonium salts as a nitrogen source can better promote the rapid removal of phenol by strain BY18.

[0017] The strain BY18 of this invention significantly improves the maximum phenol removal rate under mixed nitrogen source conditions, reaching as high as 92.52 mg / L / h.

[0018] During the simultaneous removal of phenol and nitrogen by the strain BY18 of this invention, the amount of denitrification intermediates generated is extremely small. Attached Figure Description

[0019] Figure 1 The morphology of strain BY18 is shown below; where A is the colony morphology on LB medium; B is the colony morphology on BTB plate; C is the Gram-stained colony morphology under an optical microscope; and D is the cell morphology under a scanning electron microscope.

[0020] Figure 2 Phylogenetic tree of strain BY18 based on the 16S rRNA gene sequence and other related sequences;

[0021] Figure 3 Figure showing the test results for the optimal conditions for simultaneous denitrification and phenol removal;

[0022] Figure 4 The response surface plot shows the effect of the interaction between various factors on the denitrification and phenol removal efficiency of BY18.

[0023] Figure 5 The contour plot shows the effect of the interaction between various factors on the denitrification and phenol removal efficiency of BY18. In the plot, a, b, and c represent the removal rate of ammonium nitrogen, d, e, and f represent the removal rate of phenol, and g, h, and i represent the removal rate of total nitrogen.

[0024] Figure 6 The graph shows the test results of HN-AD and phenol degradation ability of strain BY18. Among them, A is the test result using ammonium nitrogen as nitrogen source; B is the test result using nitrate nitrogen as nitrogen source; C is the test result using nitrite nitrogen as nitrogen source; and D is the test result using a mixture of nitrate nitrogen and nitrite nitrogen as nitrogen source.

[0025] Figure 7 The graphs show the SND and phenol removal capacity test results of strain BY18. Figure A shows the test results using a mixture of 75 mg / L ammonium salt and 50 mg / L nitrate as the nitrogen source; Figure B shows the test results using a mixture of 75 mg / L ammonium salt and 50 mg / L nitrite as the nitrogen source; Figure C shows the test results using a mixture of 25 mg / L ammonium salt and 25 mg / L nitrate as the nitrogen source; and Figure D shows the test results using a mixture of 25 mg / L ammonium salt and 25 mg / L nitrite as the nitrogen source. Detailed Implementation

[0026] The examples provided are for illustrative purposes only and are not intended to limit the scope of the invention. Therefore, non-essential modifications and adjustments made to the embodiments by those skilled in the art based on the above description are still within the protection scope of this invention.

[0027] culture medium

[0028] Basic culture medium (1L): 0.236g (NH4)2SO4, 0.980g C6H5OH, 0.04g MgSO4, 0.014g CaCl2, 0.009g Fe2SO4, 3.5g K2HPO4 and 1.5g KH2PO4.

[0029] Enrichment medium: The composition is the same as the basal medium, but the pH is adjusted to 7.2. This medium is used to isolate strains that are simultaneously denitrified and phenol-removed.

[0030] Bromothymol Blue (BTB) solid medium (1L): 1 mL of BTB (1.5%) anhydrous ethanol solution, 0.592 g FeSO4·7H2O, 0.488 g MgSO4, 1 g KH2PO4, 0.094 g CaCl2, 0.236 g (NH4)2SO4, 2.45 g sodium succinate, 18 g agar powder, pH 8.2. This medium was used for the isolation, purification, and morphological identification of strain BY18.

[0031] LB medium (1L): 5g yeast extract, 10g NaCl, 10g tryptone, and pH 7.2. This medium is used to activate and propagate the strain.

[0032] Nitrification medium (1L): 0.354g (NH4)2SO4, other components are the same as the basal medium;

[0033] Denitrification medium (1L): Replace (NH4)2SO4 in the basal medium with 0.246g NaNO2 or 0.361g KNO3 or 0.123g NaNO2 and 0.181g KNO3, and the other components are the same as the basal medium;

[0034] Simultaneous nitrification and denitrification medium (1L): Replace the (NH4)2SO4 in the basal medium with 0.354g (NH4)2SO4 and 0.246g NaNO2 / 0.354g (NH4)2SO4 and 0.361KNO3 / 0.118g (NH4)2SO4 and 0.123g NaNO2 / 0.118g (NH4)2SO4 and 0.181KNO3. The other components are the same as the basal medium.

[0035] The pH of the nitrification medium, denitrification medium, and SND medium was all 8.2;

[0036] All the above culture media were sterilized at 121℃ and 0.11MPa for 30 min, and phenol was added after filtration sterilization.

[0037] Separation and identification

[0038] Soil samples were collected from a Juncus effusus wet meadow in Weining County, Guizhou Province, China. Approximately 1 g of soil was transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterile enrichment medium and incubated at 25 °C and 150 r / min for 5 days. 5 mL of bacterial suspension was then transferred to fresh enrichment medium and incubated for 5 days. This operation was repeated 3 times.

[0039] Then, the bacterial suspension was serially diluted 10-10 -3 -10 -8 Take 5 μL of the diluted solution and spread it onto a plate containing BTB solid medium. Incubate at 25°C for 5 days.

[0040] The colonies that turned BTB medium blue were purified by streaking 3-5 times. The strain with the highest removal rates of para-ammonia and phenol was selected as the candidate strain and stored in 30% glycerol at -20°C. The colony morphology of the obtained strain BY18 on LB medium is shown in the figure. Figure 1 As shown in Figure A, the colony morphology on the BTB plate is as follows: Figure 1 As shown in B;

[0041] The candidate strains were Gram-stained, and the staining results were observed using an optical microscope. Bacterial morphology was also observed using a scanning electron microscope, and the results are as follows: Figure 1 C and Figure 1 As shown in D.

[0042] Depend on Figure 1 It can be seen that the colony morphology of strain BY18 on BTB solid medium is round, with a slightly raised blue center and a regular milky white edge. The surface is smooth, moist and opaque. Gram staining shows that strain BY18 is Gram negative and is short rod-shaped. Scanning electron microscopy results show that strain BY18 has raised surfaces, but no spores or flagella are seen.

[0043] DNA was extracted from strain BY18 using a bacterial DNA extraction kit (Magen). The target gene was cloned using a DNA gel recovery kit and a T-vector PCR product cloning kit (Sangon Biotech). Positive clones were amplified by PCR using M13 universal primers and then sequenced by Sangon Biotech (Shanghai, China). The obtained 16S rRNA gene sequence was submitted to the National Center for Biotechnology Information (NCBI) to obtain the strain accession number (OR616619). A phylogenetic tree was constructed using MEGA 5.0. The results are as follows: Figure 2 As shown.

[0044] Depend on Figure 2 It can be seen that strain BY18 is closely related to Acinetobacterguillouiae.

[0045] Optimal conditions for simultaneous denitrification and phenol removal

[0046] The biodegradation rates of ammonium and phenol by strain BY18 were optimized using a single-factor method. When studying the effect of a single factor, other factors were kept constant to determine and maintain the optimal level of that factor. Then, the effects of other factors were investigated. These environmental factors included temperature (10℃, 15℃, 20℃, 25℃, 30℃, 35℃), rotation speed (0, 50 r / min, 100 r / min, 150 r / min, 200 r / min), pH (5.2, 6.2, 7.2, 8.2, 9.2), C / N ratio (5, 10, 15, 20, 25, 30), and inoculum size (0.2 × 10⁻⁶). 8 CFU / mL, 0.3×10 8 CFU / mL, 0.4×10 8 CFU / mL, 0.5×10 8 CFU / mL, 0.6×10 8 CFU / mL, 0.7×10 8 The effect of CFU / mL is as follows:

[0047] Take 1 mL of the bacterial suspension preserved with glycerol and transfer it to LB liquid medium. Incubate at 25℃ and 150 r / min for 24 h. Then transfer 3.5 mL of the bacterial suspension to fresh LB liquid medium and culture for 36 h under the same conditions. Set up groups with culture temperatures of 10℃, 15℃, 20℃, 30℃ and 35℃.

[0048] After culturing, the bacterial culture was centrifuged at 6500 rpm for 5 min. The nutrient solution was discarded, and the bacterial cells were washed three times with sterile water by centrifugation. The resuspended bacterial cells were then inoculated into basal culture medium, and the cell optical density (OD) was measured after incubation for 21 h. 600 pH value, ammonium nitrogen concentration, total nitrogen concentration, and phenol concentration;

[0049] An uninoculated culture medium was used as a blank control. Three samples were prepared. The pH value was measured using a pH meter, and the phenol concentration, nitrogen concentration, and OD were measured using a spectrophotometer. 600 The values ​​were statistically analyzed and charts were created using Excel, SPSS, MEGA 5.0, and Origin 2022. All results in this application are expressed as mean ± standard deviation (SD), and the same applies below. The results are as follows. Figure 3 As shown.

[0050] Temperature not only affects the activity of related enzymes, but also influences the synthesis of substances, enzymes, and proteins both inside and outside cells (Hu, B., Lu, JY, Qin, YX, Zhou, M., Tan, Y., Wu, P., Zhao, JQ, 2023. A critical review of heterotrophic nitrification and aerobic denitrification process: Influencing factors and mechanisms. J. Water Process Eng. 54). Temperatures that are too high or too low can lead to decreased enzyme activity or even complete inactivation, thereby affecting biochemical reactions, such as bacterial metabolism of carbon and nitrogen. Figure 3As shown in Figure A, strain BY18 can grow in a temperature range of 10-35℃ and can effectively remove a certain amount of ammonium and phenol. At 25℃, strain BY18 exhibits the highest removal rates for ammonium nitrogen, total nitrogen, and phenol, at 97.02%, 90.71%, and 87.68%, respectively. There is no significant difference in the removal rates of ammonium salts and phenol between 25℃ and 30℃. However, the death and decomposition of bacterial cells release nitrogenous substances such as nucleic acids and proteins, which explains why the total nitrogen removal efficiency is only 81.01% at 30℃. Furthermore, the removal efficiency of ammonium nitrogen and phenol is lower at 35℃ than at 25℃. It is noteworthy that in previous studies, most strains exhibited strong ammonium nitrogen and phenol degradation capabilities within the 30-37°C temperature range (Rajta, A., Bhatia, R., Setia, H., Pathania P., 2020. Role of heterotrophic aerobic denitrifying Bacteria innitrate removal from wastewater. J. Appl. Microbiol. 128(5), 1261–1278). For example, strain WY-01 (Wang, Y., Chen, H., Liu, YX, Ren RP, Lv, YK, 2016. An adsorption-release-biodegradation system for simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater. Bioresource Technology. 211) almost completely degraded phenol at 30-35°C. However, when the temperature was above or below this range, the degradation efficiency decreased significantly, falling below 20%. Clearly, strain BY18 exhibits broad temperature adaptability. Temperature has a significant impact on the simultaneous removal of phenol and ammonium. The optimal temperature for the degradation of phenol and ammonium nitrogen by strain BY18 is 25℃. In subsequent studies on the optimal conditions for other parameters, the temperature was set to 25℃.

[0051] The shaking speed controls the concentration of dissolved oxygen in the culture medium, which is a prerequisite for heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria to operate in an aerobic state. As is well known, oxygen is both the electron acceptor in heterotrophic nitrification and the final electron acceptor in aerobic respiration. When the shaking speed increases from 0 to 200 r / min, the removal rates of phenol and ammonium nitrogen also increase (e.g., ...). Figure 3(As shown in B). The increased rotation speed increased the DO concentration in the culture medium and enhanced the contact between bacteria and phenol and ammonium, thus promoting the removal rates of ammonium and phenol, as well as cell growth. At a rotation speed of 150 r / min, strain BY18 achieved removal rates of 97.31%, 88.42%, and 86.97% for ammonium nitrogen, total nitrogen, and phenol within 21 hours, respectively, not significantly different from that at 200 r / min. This may be because DO primarily affects intracellular redox reactions by altering the oxidation-reduction potential (ORP) in the culture medium, and ORP is closely related to the electron transport chain (Hu, B., Lu, JY, Qin, YX, Zhou, M., Tan, Y., Wu, P., Zhao, JQ, 2023. A critical review of heterotrophicnitrification and aerobic denitrification process: Influencing factors and mechanisms. J. Water Process Eng. 54). At 200 r / min, the dissolved oxygen (DO) in the culture medium affected the electron transport chain by altering the oxygen respiration rate (ORP). However, the electron transport chain may have already reached saturation at this point, thus the removal of phenol and inorganic nitrogen did not continue to increase. Interestingly, the phenol removal rate at 200 r / min was slightly lower at 81.41%, and there was no significant difference in cell optical density compared to 150 r / min. This suggests that bacterial growth was not inhibited by high concentrations of dissolved oxygen, but rather by the large shear force generated by excessively high rotation speed, which damaged the cells (Hu, JA, Yang, XY, Deng, XY, Liu, XM, Yu, JX, Chi, R., Xiao, CQ, 2022. Isolation and nitrogen removal efficiency of the heterotrophic nitrifying-aerobic denitrifying strain K17 from a rare earthelement leaching site. Front. Microbiol., 13, 905409). Therefore, the optimal rotation speed for the growth of strain BY18 cells and the removal of ammonium and phenol was 150 r / min. In subsequent studies on the optimal conditions for other parameters, the rotation speed was set to 150 r / min.

[0052] Any change in pH affects bacterial growth and the activity of key enzymes in various metabolic processes (Zhang, MM, He, TX, Chen, MP, Wu, QF, 2022. Ammonium and hydroxylamine can be preferentially removed during simultaneous nitrification and denitrification by Pseudomonas taiwanensis EN-F2. Bioresour. Technol. 350, 126912). Furthermore, it affects bacterial cell membrane potential, directly interfering with proton synthesis and consumption, and indirectly regulating the energy yield of redox reactions (Jin, QS, Kirk, MF, 2018. pH as a primary control in environmental microbiology: 1. Thermodynamic perspective. Front. Environ. Sci. 6). For example... Figure 3As shown in Figure C, the optimal pH for strain BY18 is 7.2–8.2. Within this range, the removal rates of ammonium nitrogen, total nitrogen, and phenol are 95.65%–96.46%, 87.83%–87.94%, and 86.59%–88.30%, respectively. Furthermore, under both acidic and alkaline conditions, the phenol removal rate of strain BY18 exceeds 50%. When the pH is adjusted to 9.2, the removal rates of ammonium nitrogen and phenol reach 81.21% and 76.98%, respectively. However, studies have shown that the optimal pH for most aromatic compound degrading microorganisms is acidic or neutral (Yan et al., 2021). For example, the optimal pH for phenol degradation by Candidagenus JS3 is 6 (Jiang, Y., Yang, K., Wang, HY, Shang, Y., Yang, XJ, 2015. Characteristics of phenol degradation in saline conditions of a halophilic strain JS3 isolated from industrial activated sludge. Mar. Pollut. Bull. 99(1-2), 230–234). It is noteworthy that the small-molecule organic acids produced during phenol decomposition lower the pH of the culture medium, and a gradually increasing acidity environment will eventually lead to the death of the strain. However, in recent years, some bacteria, like strain BY18, have been found to prefer alkaline initial growth environments, such as Bacillus cereus ZWB3 (Zhang, JF, Zhou, X., Zhou, Q., Zhang, JJ, Liang, J., 2022. A study of highly efficient phenol biodegradation by a versatile Bacillus cereus ZWB3 on aerobic condition. Water Sci. Technol. 86(2), 355–366) and Acinetobacter calcoaceticus PA (Liu, ZH, Xie, WY, Li, DH, Peng, Y., Li, ZS, Liu, SS, 2016. Biodegradation of phenol by bacteria strain Acinetobacter Calcoaceticus PA isolated from phenolic wastewater. Int. J. Environ. Res. Public Health 13(3), 300).On the other hand, strain BY18 was less effective at removing ammonium salts in an acidic environment (pH 5.2) than in an alkaline environment. This difference may be due to the higher NH3 concentration in an alkaline environment. The ammonia monooxygenase responsible for deammoniation prefers NH3 as a substrate rather than NH4. + (Yang, M., Lu, DW, Qin, BD, Liu, QL, Zhao, YM, Liu, HL, Ma, J., 2018. Highly efficient nitrogen removal of a coldness-resistant and low nutrient needed bacterium, Janthinobacterium sp. M-11. Bioresour. Technol., 256, 366–373). In summary, strain BY18 showed the best performance in the optimal pH range of 7.2–8.2 for the degradation of inorganic nitrogen and phenol and for sustained cell growth. In subsequent studies on optimal conditions for other parameters, pH was set to 8.2.

[0053] Carbon sources act as electron donors in biological denitrification, and the C / N ratio is a measure of the ratio of electron donors to electron acceptors during denitrification. The ratio of intracellular electron carriers NADH and NAD+, the activity level of the electron transport chain, and ATP levels all change with variations in the C / N ratio. In this study, the C / N ratio of the culture medium was adjusted by changing the concentration of ammonium nitrogen. By maintaining a phenol concentration of 980 mg / L (with a carbon concentration of 750 mg / L), and varying the (NH4)2SO4 mass to 0.708 g / L, 0.354 g / L, 0.236 g / L, 0.177 g / L, 0.142 g / L, and 0.118 g / L, the nitrogen concentrations were changed to 150 mg / L, 75 mg / L, 50 mg / L, 37.5 mg / L, 30 mg / L, and 25 mg / L, respectively, to adjust the C / N ratio. The results are as follows: Figure 3As shown in Figure D, strain BY18 exhibited the highest phenol removal rate (94.39%) when the C / N ratio was 10. At C / N ratios of 20, 25, and 30, due to the lower substrate ammonium nitrogen concentration, strain BY18 achieved similar ammonium nitrogen removal rates (all above 99%), but showed significant differences in phenol removal rates. The same pattern was observed at C / N ratios of 10 and 15, likely because the energy obtained by bacteria from ammonium oxidation promotes phenol oxidation; higher ammonium ion concentrations result in greater phenol removal rates. However, excessively high nitrogen concentrations, i.e., low carbon levels, prevented the electron flow from meeting the energy requirements for bacterial growth. For example, at a C / N ratio of 5 (i.e., an ammonium nitrogen concentration of 150 mg / L), both inorganic nitrogen and phenol removal rates significantly decreased. In conclusion, the optimal C / N ratio for strain BY18 to simultaneously degrade phenol and ammonium is 10, and this ratio will be maintained in subsequent studies on optimal conditions for other parameters.

[0054] The initial inoculum size in the culture medium also affects bacterial carbon and nitrogen metabolism. Too low an initial inoculum size makes the bacteria less tolerant of toxic compounds in the simulated wastewater, while too high an inoculum size leads to competition for substrates and bacterial autolysis. Therefore, both excessively high and low inoculum sizes negatively impact the biodegradation of phenol and ammonium nitrogen. Figure 3 As shown in E, the optimal inoculum size for strain BY18 is 0.2 × 10⁻⁶. 8 CFU / mL. At this inoculum level, strain BY18 achieved removal rates of 99.68% for ammonium nitrogen, 88.76% for total nitrogen, and 99.63% for phenol, all higher than other inoculum levels.

[0055] Response surface optimization design

[0056] The optimal conditions for the degradation of ammonium and phenol by strain BY18 were further optimized using response surface methodology. Based on the results of single-factor experiments, temperature (A: 20℃, 25℃, and 30℃), pH (B: 6.2, 7.2, and 8.2), and rotation speed (C: 100 r / min, 150 r / min, and 200 r / min) were selected as the factors of evaluation. Ammonium removal efficiency (R1), phenol removal efficiency (R2), and total nitrogen removal efficiency (R3) were used as response values. A three-factor, three-level Box-Behnken design was used in Design Expert 12.0, and response surface analysis was performed using Design Expert 12. The results are shown in Tables 1 to 4. Figures 4 to 5 As shown.

[0057] Table 1 Experimental design and results of response surface methodology.

[0058]

[0059] Table 2. Analysis of variance of the quadratic model for ammonium nitrogen removal efficiency.

[0060] Model 10422.51 9 1158.06 33.78 <0.0001 *** A-Temperature 3902.33 1 3902.33 113.83 <0.0001 *** B-pH 4249.59 1 4249.59 123.96 <0.0001 *** C-speed 434.45 1 434.45 12.67 0.0092 ** AB 399.70 1 399.70 11.66 0.0112 * AC 116.91 1 116.91 3.41 0.1073 BC 112.75 1 112.75 3.29 0.1126 <![CDATA[A 2 ]]> 778.29 1 778.29 22.70 0.0020 ** <![CDATA[B 2 ]]> 166.15 1 166.15 4.85 0.0636 <![CDATA[C 2 ]]> 158.31 1 158.31 4.62 0.0687 residual 239.97 7 34.28 Missing item 113.18 3 37.73 1.19 0.4194 Not significant Pure error 126.80 4 31.70 sum 10662.48 16 <![CDATA[Coefficient of determination (R 2 )]]> 0.9775 <![CDATA[Adjusted R 2 > 0.9486 <![CDATA[Predicting R 2 > 0.8116

[0061] Table 3 Analysis of variance for the quadratic model of phenol removal efficiency

[0062]

[0063]

[0064] Table 4 Analysis of variance for the quadratic model of total nitrogen removal efficiency

[0065] Model 9758.22 9 1084.25 65.38 <0.0001 *** A-Temperature 2866.42 1 2866.42 172.83 <0.0001 *** B-pH 4166.38 1 4166.38 251.21 <0.0001 *** C-speed 534.96 1 534.96 32.26 0.0008 *** AB 407.49 1 407.49 24.57 0.0016 ** AC 159.69 1 159.69 9.63 0.0172 * BC 123.13 1 123.13 7.42 0.0296 * <![CDATA[A 2 ]]> 717.39 1 717.39 43.26 0.0003 *** <![CDATA[B 2 ]]> 272.50 1 272.50 16.43 0.0048 ** <![CDATA[C 2 ]]> 360.96 1 360.96 21.76 0.0023 ** residual 116.10 7 16.59 Missing item 67.60 3 22.53 1.86 0.2772 Not significant Pure error 48.49 4 12.12 sum 9874.32 16 <![CDATA[Coefficient of determination (R 2 )]]> 0.9882 <![CDATA[Adjusted R 2 > 0.9731 <![CDATA[Predict R 2 > 0.8828

[0066] Note: "*" indicates significant (0.01 < p value < 0.05), "**" indicates extremely significant (0.001 < p value < 0.01),

[0067] "***" indicates highly significant (p value < 0.001).

[0068] It can be seen from Table 1 that the relationship between each factor and the response value is expressed by the quadratic equation as follows:

[0069] Y1 = 82.17 + 22.09A + 23.05B + 7.37C - 10.00AB + 5.41AC - 5.31BC - 13.60A 2 - 6.28B 2 - 6.13C 2 ;

[0070] Y2 = 96.67 + 18.62A + 14.05B + 3.89C - 6.90AB + 4.26AC - 4.40BC - 17.90A 2 - 4.25B 2 - 9.89C 2 ;

[0071] Y3 = 77.86 + 18.93A + 22.82B + 8.18C - 10.09AB + 6.32AC - 5.55BC - 13.05A 2 - 8.04B 2 - 9.26C 2 ;

[0072] Wherein, Y1, Y2 and Y3 represent ammonium removal rate, phenol removal rate and total nitrogen removal rate respectively, and A, B and C represent temperature, pH and rotation speed respectively.

[0073] Tables 2 to 4 show that the models for all three response values ​​are highly significant (p < 0.0001), and the lack of fit is not significant (p > 0.05). This indicates that the experimental results are reliable and the models adequately fit the three response values. Furthermore, the coefficients of determination (R²) for the three models are... 2 All of them are greater than 0.97, and the predictive determination coefficient (R²) is greater than 0.97. 2 ) and the adjusted coefficient of determination (Adjusted R) 2 The differences are all less than 0.2. These results indicate that the model is reliable and can be effectively used to predict and analyze the removal efficiency of ammonium, phenol, and total nitrogen. Regarding ammonium nitrogen removal efficiency, the primary terms A, B, C, and the secondary term A... 2 The interaction between factors A and B (AB) also has a significant impact. The linear terms A, B, and C, the interaction term AB, and the quadratic term A are all significantly affected. 2 C 2 All factors significantly affected the phenol removal rate of strain BY18. Notably, all primary, secondary, and interaction terms had a significant impact on total nitrogen removal. Analysis of the F-values ​​showed that the order of influence of the three factors on the removal efficiency of ammonium nitrogen and total nitrogen was pH > temperature > DO. pH had the greatest impact, possibly because pH affects cell growth, metabolism, and the NH3 / NH4 ratio. + -N and HNO2 / NO2 — The balance (Yang, M., Lu, DW, Qin, BD, Liu, QL, Zhao, YM, Liu, HL, Ma, J., 2018. Highly efficient nitrogen removal of a coldness-resistant and low nutrient needed bacterium, Janthinobacterium sp. M-11. Bioresour. Technol., 256, 366–373). On the other hand, the order of importance of factors affecting phenol removal rate is temperature > pH > DO, that is, temperature has a greater impact on the activity of enzymes related to phenol removal.

[0074] Depend on Figure 4 and Figure 5It can be seen that with the increase of pH, temperature, and rotation speed, the removal rates of ammonium nitrogen, phenol, and total nitrogen all show a trend of first increasing and then decreasing. Furthermore, the contour plot is elliptical, indicating the interaction between the factors. Using DesignExpert 12.0 software, it was predicted that at pH 8.2, temperature 27.024℃, and rotation speed 163.966 r / min (adjusted to pH = 8.2, temperature 27℃, and rotation speed 164 r / min after feasibility consideration), the removal efficiencies of ammonium, total nitrogen, and phenol could reach their maximum values ​​of 100%, 94.8%, and 100%, respectively, after 21 h of cultivation. Three parallel experiments were conducted under the predicted conditions to verify this, and the results showed that the removal rates of ammonium nitrogen, total nitrogen, and phenol were 94.21%, 85.65%, and 97.40%, respectively, reaching the predicted values ​​of 94.21%, 90.35%, and 97.40%, indicating that the model is feasible. Single-factor experiments showed that when the pH was 8.2, the temperature was 25℃, and the rotation speed was 150 r / min, the maximum removal rates of ammonium nitrogen, total nitrogen, and phenol were 99.68%, 88.76%, and 99.63%, respectively, which were not significantly different from the actual results under the predicted conditions. Considering the operability of practical applications, the following settings were selected: pH 8.2, temperature 25℃, rotation speed 150 r / min, C / N ratio 10, and inoculum size 0.2 × 10⁻⁶. 8 Further studies will be conducted on CFU / mL.

[0075] HN-AD and phenol degradation performance tests of strain BY18

[0076] The simultaneous nitrification and phenol removal capabilities of strain BY18 were investigated using phenol and ammonium sulfate as single carbon and nitrogen sources, respectively. The initial concentrations of phenol and ammonium nitrogen were set at 980 mg / L and 75 mg / L, respectively. Strain BY18 was activated, expanded, and washed using the same method described above. An appropriate amount of bacterial suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterilized nitrification, denitrification, and SND media. The initial cell optical density (OD) of strain BY18 at 600 nm was controlled to be 0.2. The cells were incubated at 25 °C and 150 r / min, and the pH and cell optical density (OD) were measured at regular intervals. 600The concentrations of phenol and nitrogen (including ammonium nitrogen, nitrate nitrogen, nitrite nitrogen, and total nitrogen) were measured every 6 hours during nitrification, SND, and when nitrite and nitrate were used as a mixed nitrogen source, and every 9 hours during denitrification. The concentrations of ammonium, nitrate, total nitrogen, and nitrite were determined using the indophenol blue method, ultraviolet spectrophotometry, alkaline potassium persulfate digestion-ultraviolet spectrophotometry, and N-(1-naphthyl)-ethylenediamine spectrophotometry (He,TX,Wu,QF,Ding,CY,Chen,MP,Zhang,MM,2021.Hydroxylamine and nitrite are removed effectively by Streptomycesmediolani strain EM-B2.Ecotoxicol.Environ.Saf.224,112693.Advance onlinepublication.). Phenol concentration was determined by the 4-aminoantipyrine method (Shahryari, S., Zahiri, HS, Haghbeen, K., Adrian, L., Noghabi, KA, 2018. High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity. Ecotoxicol. Environ. Saf. 164, 455–466), and the results are as follows. Figure 6 As shown in A;

[0077] Using the same method, ammonium nitrogen was replaced with nitrate nitrogen, nitrite nitrogen, and a combination of nitrate and nitrite nitrogen for testing, and the results are as follows: Figure 6 As shown in B, 6C, and 6D.

[0078] Because phenol is highly toxic, most bacteria cannot survive or grow slowly in high-concentration phenol solutions, exhibiting a long lag phase. However, strain BY18 showed a lag phase of only 6 hours in a 984.14 mg / L phenol solution (e.g., ...). Figure 6As shown in A), the lag time is significantly shorter than that of most bacteria. For example, in a phenol solution with a concentration of 914.28 mg / L, the lag time of Rhodococcus sp. SKC is 42.26 h (Wen, YJ, Li, CF, Song, XM, Yang, YS, 2020. Biodegradation of phenol by Rhodococcus sp. strain SKC: characterization and kinetics study. Molecules 25(16), 3665). Acinetobacter johnsonii CFII-99A (Fikó D.R., Ráduly B., Máthé I., T.,Lányi,S.,Szilveszter,S.,2023. Bioaugmentation potential investigation using a phenol affinity analysis of three Acinetobacter strains in a multi-carbon-source condition. Water 15(15)) had a hysteresis period of 12 h in a 500 mg / L phenol solution, indicating that strain BY18 has the ability to rapidly adapt to high concentrations of phenol. In addition, strain BY18 removed 99.89% of the phenol within 18 h at a removal rate of 54.61 mg / L / h. Compared with other reports, the phenol removal rate was similar to that of Rhodococcus ruber C1 (50.17 mg / L / h) (Zhao, TT, Gao, YH, Yu, TT, Zhang, YR, Zhang, ZY, Zhang, L., Zhang, LJ, 2021. Biodegradation of phenol by a highly tolerant strain Rhodococcus ruber C1: Biochemical characterization and comparative genome analysis. Ecotoxicol. Environ. Saf. 208, 111709), but significantly higher than that of Candida genus JS3 (20.83 mg / L / h) (Jiang, Y., Yang, K., Wang, HY, Shang, Y., Yang, XJ, 2015. Characteristics of phenol degradation in saline conditions of ahalophilic strain JS3 isolated from industrial activatedsludge.Mar.Pollut.Bull.99(1-2),230–234), Acinetobacter lwoffii NL1’s 41.67 mg / L / h (Xu, N., Qiu, C., Yang, QY, Zhang, YZ, Wang, MQ, Ye, C., Guo, ML2021.,Analysis of phenol biodegradation in antibiotic and heavy metal resistant Acinetobacterlwoffii NL1.Front.Microbiol.12,725755)、Acinetobacter radioresistens 27.78mg / L / h of APH1 (Liu, YF, Wang, WW, Shah, SB, Zanaroli, G., Xu, P., Tang, HZ, 2020. Phenol biodegradation by Acinetobacter radioresistens APH1 and its application in soil bioremediation.Appl.Microbiol.Biotechnol.104(1),427–437) and 2.31mg / L / h of Sulfobacillus acidophilus TPY (Zhou, WG, Guo, WB, Zhou, HB, Chen, XH, 2016. Phenol degradation by Sulfobacillus acidophilus TPY via The results showed that strain BY18 could rapidly remove high concentrations of phenol, leading to massive cell proliferation and thus increasing the removal rates of ammonium nitrogen and total nitrogen. After 24 hours of culture, strain BY18 achieved a 94.6% removal rate of ammonium nitrogen, with the maximum removal rate occurring between 6 and 12 hours post-culture, at 4.36 mg / L / h, which was higher than that of some other bacteria that also use phenol as their sole carbon source. For example, the maximum removal rate of ammonium nitrogen by Pseudomonas sp. HJ3 in a medium containing 50 mg / L ammonium nitrogen and 200 mg / L phenol was only 2.10 mg / L / h (Ma, WW, Han, YX, Ma, WC, Han, HJ, Xu, CY, Zhu, H., 2019. Simultaneous nitrification and denitrification (SND) bioaugmentation with Pseudomonas sp. HJ3 inoculated for enhancing phenol and nitrogen removal in coal gasification wastewater. Water Sci.).Technol. 80(8), 1512–1523). Similarly, the maximum removal rates of Serratia sp. LJ-1 for ammonium nitrogen and phenol were only 1.08 mg / L / h and 2.14 mg / L / h, respectively (Lu J., Jin Q., He Y.L., He, X., Zhao, J., 2014. Simultaneous removal of phenol and ammonium using Serratia sp. LJ-1 Capable of heterotrophicnitrification-aerobic denitrification. WaterAir Soil Pollut. 225(9)). More importantly, the rate of 4.36 mg / L / h is even higher than that of Halomonas sp. DN3 (2.05 mg / L / h) (Xie, YM, Tian, ​​XL, He, Y., Dong, SL, Zhao, K., 2023. Nitrogen removal capability and mechanism of a novel heterotrophic nitrification-aerobic denitrification bacterium Halomonas sp. DN3. Bioresour. Technol. 387, 129569) and Acinetobacter indicus ZJB20129 (3.66 mg / L / h) (Ke, X., Liu, C., Tang, SQ, Guo, TT, Pan, L., Xue, YP, Zheng, YG, 2022. Characterization of Acinetobacter indicus ZJB20129 for heterotrophic nitrification and aerobic denitrification isolated from) using simple organic matter as a carbon source. an urban sewage treatment plant. Bioresour. Technol. 347, 126423) and Achromobacter denitricans QHR-5 (3.17 mg / L / h) (Mu, H., Li, WX, Hu, KY, Zhu, HJ, Ren S., Peng, YZ, Hei SL, Li, J., Quan, HR, 2023.Characterization of Achromobacter denitrificans QHR-5 for heterotrophic nitrification-aerobicdenitrification with iron oxidation function isolated from BSIS: Nitrogen removal performance and enhanced SND capability of BSIS. (Biochem. Eng. J. 191). Indeed, simple organic compounds are more effective in promoting cell growth and removing ammonium nitrogen because they are non-toxic and have no harmful effects on bacteria. Furthermore, their simple molecular structures make them more readily involved in the TCA cycle of bacterial carbon metabolism. This result indicates that strain BY18 has an advantage in removing high concentrations of phenol and ammonium nitrogen. In particular, no intermediate products such as hydroxylamine, nitrate, and nitrite were detected during nitrification. However, in the strain Pseudomonas sp.HJ3(Ma,WW,Han,YX,Ma,WC,Han,HJ,Xu,CY,Zhu,H.,2019.Simultaneousnitrification and denitrification(SND)bioaugmentation withPseudomonas sp.HJ3inoculated for enhancing phenol and nitrogen removal in coal gasificationwastewater.Water Sci.Technol.80(8),1512–1523), Serratia sp.LJ-1(Lu J., Jin Q., He YL, He, Pollut.225(9)) and Diaphorobacter sp.PD-7(GeQ.L.,Yue XP, Wang GY, 2015.Simultaneous heterotrophic nitrification and aerobic denitrification at high initial phenol concentration by isolated *Diaphorobacterium diaphorobacter* sp. PD-7. *Chin. J. Chem. Eng. 23(05): 835-841* showed that intermediate products nitrate and nitrite were detected during the simultaneous nitrification and phenol removal process. Furthermore, strain BY18 achieved a total nitrogen removal rate of 90.18%, with a maximum removal rate of 4.33 mg / L / h. These results indicate that ammonium is likely removed during phenol metabolism by strain BY18 through conversion to N2O and N2, demonstrating the considerable potential of strain BY18 in treating inorganic nitrogen and phenol-contaminated wastewater.

[0079] Currently, few studies have focused on the simultaneous removal of nitrates or nitrites and phenols by bacterial strains. However, nitrates and nitrites, as intermediate products of ammonium oxidation and pollutants emitted in industrial production, often coexist with phenol in industrial wastewater from coal gasification, papermaking, coking, etc. (Ma,WW,Han,YX,Ma,WC,Han,HJ,Xu,CY,Zhu,H.,2019.Simultaneous nitrification and denitrification (SND) bioaugmentation with Pseudomonas sp.HJ3 inoculated for enhancing phenol and nitrogen removal in coal gasification wastewater.Water Sci.Technol.80(8),1512–1523)(Yan,JW,Su,HT,Kuang,XX,Luo,LX,Zhou,W.,Zhou,SQ,2021.Characteristics andmechanism of simultaneous nitrate and phenol removal by a newly isolatedCupriavidus oxalaticus T2.Int.Biodeterior.Biodegrad.161). Excessive nitrates and nitrites pose a significant threat to human health, plant and animal survival, and the entire ecosystem. This application investigates the aerobic denitrification capacity of strain BY18 using KNO3 or NaNO2 as the sole nitrogen source and phenol as the sole carbon source. Figure 6 As shown in B, OD 600 The value increased most rapidly between 18 and 27 hours of cultivation, and the removal rates of phenol and nitrate were also the highest during this period, at 68.22 mg / L / h and 2.91 mg / L / h, respectively. This indicates that strain BY18 can effectively utilize phenol to provide energy and electrons for autogenous growth and denitrification processes. Within 36 hours, the removal rates of phenol and nitrate were 99.73% (27.19 mg / L / h) and 96.23% (1.37 mg / L / h), respectively. The removal rate was slightly lower than that of Rhodococcus pyridinivorans PDB9TN-1 (28.57 mg phenol / L / h), which also used nitrate as a nitrogen source (Barik, M., Das, CP, Kumar Verma, A., Sahoo S., Sahoo NK, 2021. Metabolic profiling of phenol biodegradation by anindigenous Rhodococcus pyridinivorans strain PDB9T N-1 isolated from paperpulp wastewater. Int. Biodeterior. Biodegrad. 158, 105168), but significantly higher than that of Rhodococcus aetherivorans UCMAc-602 (18.2 mg phenol / L / h) (Nogina, T., Fomina, M., Dumanskaya, T., Zelena, L., Khomenko, L., Mikhalovsky, S., Podgorskyi, V., Gadd, GM, 2020. A newRhodococcus aetherivorans strain isolated from lubricant-contaminated soil asa prospective phenol-biodegrading agent.Appl.Microbiol.Biotechnol.104(8),3611–3625), Cupriavidus oxalaticus T2 6.89mg phenol / L / h and 0.48mgNO3 --N / L / h(Yan,JW,Su,HT,Kuang,XX,Luo,LX,Zhou,W.,Zhou,SQ,2021.Characteristics and mechanisms of simultaneous nitrate and phenol removal by a newly isolated Cupriavidus oxalaticus T2.Int.Biodeterior.Biodegrad.161), and Diaphorobactersp.PD-7 of 16.47mg phenol / L / h and 1.16mg NO3 - -N / L / h(Ge QL, Yue XP, Wang GY, 2015. Simultaneous heterotrophic nitrification and aerobic denitrification at high initial phenol concentration by isolated bacterium Diaphorobacter sp. PD-7. Chin. J. Chem. Eng. 23(05): 835-841). This means that strain BY18 is more suitable than the strains mentioned above for removing high concentrations of phenol and nitrate. Furthermore, no accumulation of nitrite and ammonium nitrogen was detected throughout the denitrification process, possibly because the intermediate products were never present or were immediately utilized by highly active enzymes after formation.

[0080] like Figure 6As shown in Figure C, strain BY18 removed 99.71% of nitrite and 91.44% of total nitrogen within 27 h at removal rates of 2.06 mg / L / h and 1.92 mg / L / h, respectively, and completely removed 979.71 mg / L of phenol within 36 h at a rate of 27.21 mg / L / h. The removal capacity of strain BY18 for both phenol and nitrite was significantly stronger than that of Serratia sp. LJ-1 (Lu J., Jin Q., He YL, He, X., Zhao, J., 2014. Simultaneous removal of phenol and ammonium using Serratia sp. LJ-1 Capable of heterotrophic nitrification-aerobic denitrification. Water Air Soil Pollut. 225(9)), the latter of which had removal rates of 0.4 mg / L / h for total nitrogen and 1.39 mg / L / h for phenol. Analysis of the maximum nitrite removal rate showed that BY18 had a maximum removal rate of 4.72 mg / L / h, which was much higher than that of Pseudomonas sp. HJ3 (Ma,WW,Han,YX,Ma,WC,Han,HJ,Xu,CY,Zhu,H.,2019.Simultaneous nitrification and denitrification (SND)bioaugmentation with Pseudomonas sp.HJ3 inoculated for enhancing phenol and nitrogen removal in coal gasification wastewater.Water Sci.Technol.80(8),1512–1523), and even higher than that of many HN-AD strains that use simple organic matter as a carbon source.For example, the maximum removal rates of nitrite by Pseudomonas sp. B-1 (Lan,MC,Yin,QD,Wang,JX,Li,M.,Li,Y.,Li,BA,2023.Heterotrophic nitrification-aerobic denitrification performance of anovel strain,Pseudomonas sp.B-1,isolated from membrane aerated biofilmreactor.Environ.Res.220,115199) and Halomonas sp.DN3 (Xie,YM,Tian,XL,He,Y.,Dong,SL,Zhao,K.,2023.Nitrogen removal capability and mechanism of a novel heterotrophic nitrification-aerobic denitrification bacterium Halomonassp.DN3.Bioresour.Technol.387,129569) were 3.92 mg / L / h and 1.32 mg / L / h, respectively. No accumulation of ammonium nitrogen or nitrate nitrogen was detected throughout the denitrification process, indicating that strain BY18 has the ability to remove nitrite using high concentrations of phenol through aerobic denitrification. This is in contrast to 1.37 mg NO3 using nitrate as the sole nitrogen source. - At nitrogen sources of -N / L / h and 1.41 mg TN / L / h, nitrite showed higher removal rates when used as the sole nitrogen source, but the phenol removal rates did not differ significantly between the two. This suggests that under these culture conditions, nitrite has a stronger electron-acquiring ability than nitrate, but requires fewer electrons. However, compared to nitrification using ammonium as the nitrogen source, denitrification showed slightly lower nitrogen and phenol removal rates. This may be because NH4+... + The nitrification process provided the strain with additional energy besides phenol oxidation, further promoting NH4+ oxidation. + The oxidation and degradation of phenol are observed. However, by extending the culture time, denitrification using nitrate or nitrite as nitrogen sources can also achieve complete removal of nitrogen pollutants and phenol. These results indicate that strain BY18 can efficiently remove phenol while simultaneously performing heterotrophic nitrification and aerobic denitrification, with nitrification capacity being stronger than denitrification capacity.

[0081] Previous studies have shown that most bacteria produce nitrite during nitrate metabolism, but no nitrite formation was detected in this study. Nitrite is known to be toxic to blood oxygen transport in organisms (Wu, QF, He, TX, Chen, MP, Zhang, MM, 2022. Nitrogen removal characterization and functional enzymes identification of a hypothermia bacterium Pseudomonasfragi EH-H1. Bioresour. Technol. 365, 128156). To investigate whether nitrite affects nitrate metabolism in strain BY18, these two inorganic nitrogen sources were used as a mixed nitrogen source, and the removal rates of phenol, nitrate nitrogen, nitrite nitrogen, and total nitrogen were measured. The results are as follows: Figure 6 As shown in D. During the first 6 hours of culture, OD... 600 The value decreased from 0.2 to 0.13, which may be due to the strong toxicity of substances in the culture system, leading to the death of some bacterial cells. After continued culturing, the bacterial OD... 600 The value gradually increased, reaching 1.09 after 36 hours of cultivation, which was consistent with strain BY18 in a single nitrate reduction system (OD). 600 The value is 1.02) and the single nitrite reduction system (OD) 600The growth difference was not significant (value 1.12). This indicates that the addition of nitrite in the nitrate reduction system had no adverse effect on the growth of strain BY18. After 30 h, the removal rates of nitrite, nitrate, total nitrogen, and phenol were 100%, 56.42%, 73.93%, and 83.18%, respectively. After 36 h of cultivation, nitrate and phenol were completely removed, and the total nitrogen removal rate reached 93.26%. Compared with the single nitrogen source culture system, the maximum removal rates of nitrite and nitrate decreased from 4.72 mg / L / h and 2.91 mg / L / h to 2.58 mg / L / h and 2 mg / L / h, respectively. The maximum total nitrogen removal rate of 4.53 mg / L / h was similar to that of the single nitrite system (4.37 mg / L / h), but significantly higher than that of the nitrate system (3.12 mg / L / h). This is consistent with the phenomenon observed in *Pseudomonas taiwanensis* EN-F2 (Zhang, MM, He, TX, Chen, MP, Wu, QF, 2022. Ammonium and hydroxylamine can be preferentially removed during simultaneous nitrification and denitrification by *Pseudomonas taiwanensis* EN-F2. Bioresour. Technol. 350, 126912). These results indicate that in a mixed nitrogen source aerobic denitrification system, strain BY18 preferentially reduces nitrite. However, nitrite and nitrate compete for electrons, inhibiting each other's reduction rates, but complete removal of both can be achieved by extending the incubation time. Since the C / N ratio remained constant, the phenol removal rate was almost unaffected.

[0082] High-concentration phenol degradation and SND ability test

[0083] Inorganic nitrogen pollution in water bodies usually exists in multiple nitrogen forms. Therefore, it is essential to determine the purification capacity of strain BY18 for mixed nitrogen sources and phenol-contaminated wastewater. Using phenol as a single carbon source, the SND (Single Nitrification and Denitrification) capacity of strain BY18 was investigated. Specifically, unlike the assessment of strain BY18's ability to simultaneously remove phenol through nitrification and denitrification, the initial concentration of phenol was set at 980.64 mg / L, and a mixture of 75 mg / L ammonium salt and 50 mg / L nitrate was used as the nitrogen source. The results are as follows: Figure 7 As shown in A;

[0084] Using the same method, the nitrogen source was replaced with a mixture of 75 mg / L ammonium salt and 50 mg / L nitrite, and the results were as follows. Figure 7 As shown in B.

[0085] like Figure 7As shown in Figure A, strain BY18 removed 96.11% of the initial concentration of phenol (980.64 mg / L) within 18 hours at a rate of 52.36 mg / L / h. The maximum removal rate of phenol was significantly increased to 74.55 mg / L / h under a mixed nitrogen source compared to a single nitrogen source. This phenomenon was also observed in Bacillus cereus ZWB3 (Zhang, JF, Zhou, X., Zhou, Q., Zhang, JJ, Liang, J., 2022. A study of highly efficient phenol biodegradation by a versatile Bacillus cereus ZWB3 on aerobic condition. Water Sci. Technol. 86(2), 355–366). The removal rate of 52.36 mg / L / h is comparable to 54.61 mg / L / h in heterotrophic nitrification, but significantly higher than the 27.19 mg / L / h achieved with nitrate as the sole nitrogen source. Therefore, it can be inferred that the combination of ammonium and nitrate may stimulate the activity of phenol hydroxylase and catechol dioxygenase, thereby increasing the rate of phenol degradation.In addition, Bacillus cereus ZWB3 (Zhang, JF, Zhou, PA (Liu, ZH, Xie, WY, Li, DH, Peng, Y., Li, ZS, Liu, SS, 2016. Biodegradation of phenol by bacteria strain Acinetobacter Calcoaceticus PA isolated from phenolic wastewater. Int. J. Environ. Res. Public Health 13(3), 300) and Pseudomonas The phenol removal rates of strain *Cepacia cepacia* (Arutchelvan, V., Kanakasabai, V., Nagarajan, S., Muralikrishnan, V., 2005. Isolation and identification of novel high strength phenol degrading bacterial strains from phenol-formaldehyde resin manufacturing industrial wastewater. J. Hazard. Mater. 127(1-3), 238–243) using ammonium nitrogen and nitrate as mixed nitrogen sources were 33 mg / L / h, 15.27 mg / L / h, and 17.36 mg / L / h, respectively, all significantly lower than the phenol removal rate of strain BY18 under the same nitrogen source conditions. On the other hand, strain BY18 achieved removal rates of 99.42% and 50.57% for 75.56 mg / L ammonium nitrogen and 50.98 mg / L nitrate nitrogen, respectively, with removal rates of 4.17 mg / L / h and 1.43 mg / L / h.Compared to nitrification and denitrification systems alone, the maximum removal rates of ammonium nitrogen and nitrate nitrogen increased from 4.36 mg / L / h and 2.91 mg / L / h to 5.7 mg / L / h and 3.78 mg / L / h, respectively, which is contrary to the results for Pseudomonas fragi EH-H1 (Wu, QF, He, TX, Chen, MP, Zhang, MM, 2022. Nitrogen removal characterization and functional enzymes identification of a hypothermiabacterium Pseudomonas fragi EH-H1. Bioresour. Technol. 365, 128156). Ammonium was preferentially utilized in the SND process of strain BY18, similar to most HN-AD strains.Such as Pseudomonas taiwanensis EN-F2 (Zhang, MM, He, TX, Chen, MP, Wu, QF, 2022. Ammonium and hydroxylamine can be preferentially removed during simultaneous nitrification and denitrification by Pseudomonas taiwanensis EN-F2. Bioresour. Technol. 350, 126912), Pseudomonas sp. B-1 (Lan, MC, Yin, QD, Wang, JX, Li, M., Li, Y., Li, BA, 2023. Heterotrophicnitrification-aerobic denitrification performance of a novel strain, Pseudomonas sp. B-1, isolated from membrane aerated biofilmreactor.Environ.Res.220,115199) and Acinetobacter sp. ND7 (Xia, L., Li, XM, Fan, WH, Wang, JL, 2020. Heterotrophic nitrification and aerobic denitrification by a novel Acinetobacter sp. ND7 isolated from municipal activated sludge. Bioresour. Technol. 301, 122749). This may be because ammonium nitrogen can be directly utilized by microorganisms through assimilation into biomacromolecules (Huang, XJ, Jiang, DH, Ni, JP, Xie, DT, Li, ZL, 2021. Removal of ammonium and nitrate by the hypothermia bacterium Pseudomonasputida Y-9 mainly through assimilation. Environ. Technol. Innovation 2021, 22(22)). For strain BY18, the presence of ammonium and nitrate nitrogen facilitated the removal of each other.This differs from the case of Pseudomonasputida Y-9 (Huang,XJ,Jiang,DH,Ni,JP,Xie,DT,Li,ZL,2021.Removal of ammonium and nitrate by the hypothermia bacterium Pseudomonasputida Y-9 mainly through assimilation.Environ.Technol.Innovation 2021,22(22)), where nitrate inhibits the removal of ammonium nitrogen. This differs from the result in Bacillus subtilis H1 (Xie,YM,Tian,XL,Liu,Y.,Zhao,K.,Li,YM,Luo,K.,Wang,B.,Dong,SL,2023.Nitrogen removal capability and mechanism of anovel heterotrophic nitrifying-aerobic denitrifying strain H1 as a potential candidate in mariculture wastewater treatment.Environ.Sci.Pollut.Res.30(48),106366–106377), where ammonium promotes nitrate removal but its own removal is inhibited by nitrate. Furthermore, the SND ability of strain BY18, which uses phenol as a carbon source, is stronger than that of some strains that use simple organic matter as a carbon source. For example, Acinetobacter indicus ZJB2019 (Ke,X.,Liu,C.,Tang,SQ,Guo,TT,Pan,L.,Xue,YP,Zheng,YG,2022.Characterization of Acinetobacter indicus ZJB20129 forheterotrophic nitrification and aerobic denitrification isolated from an urban sewage treatment plant. Bioresour.Technol.347,126423) showed maximum removal rates of only 1.20 mg / L / h for 47.7 mg / L ammonium nitrogen and 2.82 mg / L / h for 53.3 mg / L nitrate. In contrast, strain BY18 showed a nitrite accumulation of less than 0.35 mg / L throughout the entire SND process.After 36 hours of incubation, nitrates were not completely removed, with a removal rate of only 55.83%. This may be related to the oxidation of ammonium to generate nitrates, which resulted in a low TN removal rate of 53.36%.

[0086] like Figure 7As shown in Figure B, when ammonium nitrogen and nitrite are used as a mixed nitrogen source, phenol can be completely removed within 24 hours, with a removal rate of 40.76 mg / L / h. This removal rate is significantly lower than that of ammonium as the sole nitrogen source (54.61 mg / L / h) and the mixed nitrogen source of ammonium and nitrate (52.36 mg / L / h), but higher than that of nitrite as the nitrogen source (27.21 mg / L / h). The results indicate that the addition of ammonium salts can promote the removal of phenol. However, after the addition of nitrite, the removal efficiency and maximum removal rate of ammonium nitrogen both decreased significantly, to 85.20% and 4.02 mg / L / h, respectively. This is consistent with the finding that nitrite inhibits ammonium nitrogen removal in *Pseudomonas taiwanensis* EN-F2 (Zhang, MM, He, TX, Chen, MP, Wu, QF, 2022. Ammonium and hydroxylamine can be preferentially removed during simultaneous nitrification and denitrification by *Pseudomonas taiwanensis* EN-F2. Bioresour. Technol. 350, 126912). The maximum ammonium nitrogen removal rate of 4.02 mg / L / h was lower than the 5.7 mg / L / h after the addition of nitrate. The conclusion that nitrite is a more effective promoter of ammonium nitrogen oxidation than nitrate in *Pseudomonas fragi* EH-H1 (Wu,QF,He,TX,Chen,MP,Zhang,MM,2022. Nitrogen removal characterization and functional enzymes identification of a hypothermia bacterium *Pseudomonas fragi* EH-H1. Bioresour. Technol.365,128156) differs from that of nitrite. Furthermore, the removal rate and maximum removal rate of nitrite decreased to 12.75% and 0.45 mg / L / h, respectively.This means that the presence of ammonium nitrogen also hinders nitrite removal, which contradicts the observation that ammonium salts promote nitrite removal in Achromobacter denitrificans QHR-5 (Mu,H.,Li,WX,Hu,KY,Zhu,HJ,Ren S.,Peng,YZ,Hei SL,Li,J.,Quan,HR,2023.Characterization of Achromobacterdenitrificans QHR-5 for heterotrophic nitrification-aerobic denitrification with iron oxidation function isolated from BSIS: Nitrogen removal performance and enhanced SND capability of BSIS.Biochem.Eng.J.191), but is consistent with the results of Pseudomonas putida Y-9 (Huang,XJ,Jiang,DH,Ni,JP,Xie,DT,Li,ZL,2021.Removal of ammonium and nitrate by the hypothermia bacterium Pseudomonas putida). Y-9 mainly through assimilation.Environ.Technol.Innovation 2021,22(22)).

[0087] To investigate why strain BY18 could not completely remove the aforementioned mixed nitrogen source, and to determine whether it could completely remove low-concentration mixed nitrogen sources, 25 mg / L ammonium salt and 25 mg / L nitrate, and 25 mg / L ammonium salt and 25 mg / L nitrite were used as nitrogen sources, with phenol as the single carbon source. The SND capacity, TN removal rate, and intermediate products of strain BY18 were measured, and the corresponding results are as follows: Figure 7 As shown in C and 7D. Figure 7As shown in C and 7D, in both mixed nitrogen source combinations, the removal rate of ammonium nitrogen reached 99% within 12 hours, and the removal rates of nitrate nitrogen and nitrite reached 100% after 30 hours of cultivation, with the total nitrogen removal rate exceeding 91%. This indicates that in the high-concentration mixed nitrogen source system, the depletion of nutrients in the culture medium limited the oxidation capacity of strain BY18 for ammonium nitrogen and its reduction capacity for nitrate and nitrite. Notably, in the low-concentration mixed nitrogen source system, the maximum removal rate of phenol reached as high as 92.53 mg / L / h (ammonium and nitrate) and 83.16 mg / L / h (ammonium and nitrite), indicating that the C / N ratio and nitrogen source type have a significant impact on the phenol removal rate. All these results demonstrate that strain BY18 possesses highly efficient SND and phenol degradation capabilities and can be used for the treatment of various types of inorganic nitrogen and phenol-contaminated wastewater.

[0088] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A strain, characterized in that the strain is (Acinetobacter gaillouiae) BY18, preservation number CCTCC NO: M 20232439, and the maximum removal rates of the strain are as follows: ammonium nitrogen and phenol: 4.36 mg / L / h and 62.20 mg / L / h; nitrate and phenol: 2.91 mg / L / h and 68.22 mg / L / h; nitrite and phenol: 4.72 mg / L / h and 60.82 mg / L / h.

2. The application of the strain described in claim 1 in the treatment of phenol-containing and / or nitrogen-containing wastewater.

3. The use of the strain of claim 1 in the preparation of products for treating phenol-containing and / or nitrogen-containing wastewater.

4. A water treatment agent, characterized in that, The water treatment agent includes the strain described in claim 1.

5. A wastewater treatment method, characterized in that, The wastewater treatment method includes: inoculating the strain as described in claim 1 into the wastewater to be treated and culturing it.

6. The wastewater treatment method as described in claim 5, characterized in that, The inoculum size of the strain was 0.2*10⁻⁶. 8 CFU / mL -0.7*10 8 CFU / mL.

7. The wastewater treatment method as described in claim 5, characterized in that, The culture temperature is 20-30℃, and the pH is 6.2-8.2.

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