Bacterial strain composition for treating toilet sewage and application thereof

Abstract

The invention relates to a bacterial strain composition for treating toilet sewage and application of the bacterial strain composition, the bacterial strain composition for treating toilet sewage comprises a Pseudomonas tawanensis EN-F2 bacterial strain, an Acinetobacter jonsonii EN-J1 bacterial strain, a Glutinobacter arisenensis EM-H8 bacterial strain, a Leucobacter denitrificans FST-3 bacterial strain, a Providencia husteriensis FST-4 bacterial strain, a Comamonas testosterone FST-9 bacterial strain and a Proteus cibi FST-12 bacterial strain, the preservation number of the Pseudomonas tawanensis EN-F2 bacterial strain is CCTCC NO: M 20211515, and the preservation number of Acinet is CCTCC NO: M 20211515. The preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 20241977, the preservation number of the strain is CCTCC NO: M 20241978, the preservation number of the strain is CCTCC NO: M 20241978, the preservation number of the strain is CCTCC NO: M 20241975, the preservation number of the strain is CCTCC NO: M 20241976, the preservation number of the strain is CCTCC NO: M 20241976, the preservation number of the strain is CCTCC NO: M 20241977, the preservation number of the strain is CCTCC NO: M 20241977, the preservation number of the strain is CCTCC NO: M 20241977, and the preservation number of the strain is CCTCC NO: M 20241977. The strain composition disclosed by the invention has the capability of efficiently degrading NH4 < + >-N, NO3 <->-N and COD (Chemical Oxygen Demand).

Description

Technical Field

[0001] The present application relates to the technical field of sewage treatment, and in particular to a strain composition for treating toilet sewage and its application. Background Art

[0002] Toilet sewage refers to the washing water discharged by residents in their daily lives. Specifically, toilet sewage is wastewater formed by human feces, urine and toilet flushing water. Its main components are high-concentration organic matter, nitrogen, phosphorus, potassium and other nutrients. There are also a large number of pathogenic microorganisms in toilet sewage that can cause human diseases, such as fecal coliforms, Campylobacter, Vibrio cholerae, Shigella, Salmonella, etc. These pathogenic microorganisms can be transmitted through flies, water, soil, air and other media, thereby contaminating water and food. Studies have shown that improper treatment of toilet sewage may also lead to environmental problems such as eutrophication of water bodies, soil acidification and air pollution. It is reported that the amount of black water discharged every day in the world is huge. However, due to the imperfect sewage treatment facilities, most of the black water is discharged directly.

[0003] Removing excess organic matter, nitrogen and phosphorus has become an urgent problem to be solved in toilet sewage treatment. The current methods used for toilet sewage treatment include electrochemical treatment, guanidinite precipitation, solar energy and advanced oxidation. However, the above methods have high requirements for equipment and require the addition of chemical reagents. The additional chemical reagents may cause secondary pollution and increase the treatment cost. Biological treatment has the advantages of being simple, efficient and free of secondary pollution, and has been widely used in sewage treatment. In the biological treatment process, the removal efficiency of pollutants is closely related to highly active degradation microorganisms. Studies have found that after adding a single bacterium to wastewater, pollutants can be directly degraded by the added bacteria. In addition, the addition of a single bacterium can change the diversity of the microbial community structure in the water body and indirectly accelerate the removal of pollutants. For example, the strain Olivabacter jilunii PAO-9, after being added to aquaculture wastewater, not only changed the microbial community structure in the water body, but also significantly improved the removal rate of TN, TP and COD in sewage. However, when a single strain is directly added to wastewater, different strains have different adaptability to the environment, which makes the stability of a single bacterium susceptible to substrate conditions. For example, when COD concentration increased from 200 mg / L to 800 mg / L, the removal efficiency of Chelatococcus sp.Y1 for TN decreased significantly from 94.7% to 68.1%. In addition, the competition of local microorganisms in wastewater can also weaken the ability of single strains. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the present invention provides a strain composition and application thereof.

[0005] To achieve the above objectives, the scheme of this application is as follows:

[0006] In a first aspect, the present invention provides a strain composition, including Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain;

[0007] The Pseudomonas taiwanensis EN-F2 strain was deposited in the China Center for Type Culture Collection on November 30, 2021, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number of CCTCC NO: M20211515;

[0008] The Acinetobacter johnsonii EN-J1 strain was deposited in China Center for Type Culture Collection on May 23, 2022, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M2022702;

[0009] The Glutanibacter arilaitensis EM-H8 strain was deposited in China Center for Type Culture Collection on September 13, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M20241974;

[0010] The Leucobacter denitrificans FST-3 strain was deposited in the China Center for Type Culture Collection on September 13, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M20241977;

[0011] The Providencia huaxiensis FST-4 strain was deposited in the China Center for Type Culture Collection on September 13, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M20241978;

[0012] The Comamonas testosteroni FST-9 strain was deposited in the China Center for Type Culture Collection on September 13, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number of CCTCC NO: M20241975;

[0013] The Proteus cibi FST-12 strain was deposited in the China Center for Type Culture Collection on September 13, 2024, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M 20241976.

[0014] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 0.5-3.5:1.5-3.5:0.5-2.5:0.5-3.5:0.5-2.5:1.5-2.5:0.5-1.5.

[0015] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 1-3:2-3:1-2:1-3:1-2:1.5-2.5:0.5-1.5.

[0016] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 2.5-3.5: 1.5-2.5: 1.5-2.5: 0.5-1.5: 0.5-1.5: 1.5-2.5: 0.5-1.5.

[0017] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsoniiEN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 3:2:2:1:1:2:1.

[0018] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 0.5-1.5:2.5-3.5:0.5-1.5:2.5-3.5:1.5-2.5:1.5-2.5:0.5-1.5.

[0019] Optionally, the colony ratio of the Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsoniiEN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi FST-12 strain is 1:3:1:3:2:2:1.

[0020] In a second aspect, the present application provides the use of the strain composition as described above in treating toilet sewage.

[0021] In a third aspect, the present application provides a toilet sewage treatment agent, comprising the strain composition as described above.

[0022] Beneficial effects of the present invention:

[0023] In this application, four highly efficient denitrification and carbon removal bacteria isolated and screened from actual toilet sewage were combined with highly efficient denitrification and denitrification bacteria obtained in the laboratory in the early stage to construct a microbial agent FST, which has the ability to efficiently degrade NH4 + -N、NO3 - -N and COD. The nitrogen balance results showed that 47.73% of the initial ammonium nitrogen could be converted into nitrogen-containing gas through heterotrophic nitrification, and no accumulation of intermediate nitrite was detected during the removal of ammonium nitrogen by the agent; when the agent FST performed aerobic nitrification, the conversion rate of gaseous nitrogen could reach 59.33%. In addition, after adding the microbial agent FST to the actual unsterilized toilet sewage, the removal rates of ammonium nitrogen, total nitrogen and total phosphorus increased by 28.14%, 11.37% and 12.68% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The test results of strain screening process, 1A is the test results of the removal efficiency of each strain for ammonium nitrogen and COD, 1B is the test results of the removal efficiency of each strain for nitrate and COD, Removal efficiency means removal efficiency, the same below;

[0025] Figure 2 Phylogenetic tree of candidate strains;

[0026] Figure 3Figure 3A shows the test results of the effect of different culture conditions on the removal of ammonium nitrogen and COD by the bacterial agent FST (mean ± SD (standard error) of three replicates). Figures 3A, 3B and 3C show the test results of the effect of different temperatures, rotation speeds and pH values ​​on the removal of ammonium nitrogen and COD by the bacterial agent FST, respectively. Temperature indicates temperature, Shaking speed indicates rotation speed, and Nitrate concertration indicates nitrate concentration.

[0027] Figure 4 A is the test result diagram of heterotrophic nitrification and aerobic denitrification characteristics of the bacterial agent FST (average value of three replicates ± SD (standard error)), A is the test result diagram of ammonium nitrogen removal characteristics, B is the test result diagram of nitrate removal characteristics, and the horizontal axis is the culture time;

[0028] Figure 5 Figure 1 is the test result diagram of the removal characteristics of different concentrations of ammonium nitrogen and total phosphorus by the bacterial agent FST (mean ± SD (standard error) of three replicates), A is the test result diagram of the removal characteristics of different concentrations of ammonium nitrogen; B is the test result diagram of the removal characteristics of different concentrations of total phosphorus, different letters indicate significant differences between treatments (P<0.05);

[0029] Figure 6 The figures are the test results of the denitrification and phosphorus removal from toilet sewage by the bacterial agent FST (the average value ± SD of three replicates). A is the test result diagram for the removal characteristics of ammonium nitrogen, B is the test result diagram for the removal characteristics of nitrate, C is the test result diagram for the removal characteristics of total nitrogen, and D is the test result diagram for the removal characteristics of total phosphorus. C1 represents toilet sewage, C2 represents toilet sewage inoculated with the bacterial agent FST, C3 represents toilet sewage with the addition of carbon source; C4 represents toilet sewage with both toilet sewage added and inoculated with the bacterial agent FST. Different letters indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0030] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0031] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0032] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0033] (I) Screening of strains

[0034] Toilet sewage used for strain screening was collected from Qianxi County, Guizhou Province. The collected toilet sewage was added to the filter-sterilized Luria-Bertani medium and cultured at 25°C and 150r / min for 3 days, with 3 transfers every 3 days;

[0035] Then, 25ul of the enriched culture obtained in the LB liquid medium was added to Luria-Bertani (LB) solid medium, bromothymol blue (BTB) solid medium, denitrification solid medium and carboxymethyl cellulose (CMC) solid medium, and cultured in an incubator at 25°C for 24h. After the strain grew, it was streaked and purified using the culture medium. After purification, the isolated and screened single strain was preserved in 60% glycerol and placed in a refrigerator for use.

[0036] After preliminary screening, 15 single strains were obtained. Since toilet sewage contains not only a large amount of ammonium nitrogen, but also a small amount of nitrate, when exploring the denitrification ability of the strain, whether the strain has denitrification ability is also an important indicator of whether it can be used as a candidate strain. After 48 hours of cultivation, the removal rate of ammonium nitrogen (determined by ultraviolet spectrophotometry) and nitrate (determined by ultraviolet spectrophotometry) of each strain was tested. The results are as follows: Figure 1 As shown in A.

[0037] Depend on Figure 1 As shown in A, except for strain FST-13, the removal rates of ammonium nitrogen by the other strains were all above 90%, and the COD removal rates by the bacteria were all above 70%.

[0038] Similarly, a single strain was inoculated into the denitrification medium and cultured at 25°C and 150 r / min for 48 h. The removal efficiency of nitrate and COD by each strain was tested. The results are as follows: Figure 1 As shown in B.

[0039] like Figure 1As shown in B, among the 15 strains, FST-15, FST-14, FST-9, FST-12, FST-3 and FST-4 strains showed strong denitrification ability, and their nitrate removal rates from high to low were 91.15%, 78.59%, 75.33%, 74.34%, 71.7% and 53.84%, respectively.

[0040] The six strains, FST-15, FST-14, FST-9, FST-12, FST-3 and FST-4, were identified for species. DNA of the candidate strains was extracted and the amplified PCR products were entrusted to Shanghai Sangon Biotech Co., Ltd. for 16S rRNA sequencing. The GenBank BLAST program on NCBI (http: / / www.ncbi.nlm.nih.gov / Blast.cgi) was used to analyze the obtained 16S rRNA gene sequence (about 1450 bp). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA5 software program. The results are shown in the figure. Figure 2 shown.

[0041] After identification, strains FST-14 and FST-15 were pathogenic bacteria. Therefore, the present application selected strains FST-9, FST-12, FST-3 and FST-4 as candidate strains to construct microbial agent FST. And strains FST-9, FST-12, FST-3 and FST-4 were identified as Comamonas testosteroni, Proteus cibi, Leucobacter denitrificans and Providencia huaxiensis, respectively, and their accession numbers in the GenBank database are shown in Table 1.

[0042] Table 1 Accession numbers of strains in the GenBank database

[0043] Strain name Species Accession Number FST-3 Leucobacter denitrificans PP717005 FST-4 Providencia huaxiensis PP717006 FST-9 Comamonas testosteroni PP717008 FST-12 Proteus cibi PP7170010

[0044] (II) Optimization of strain ratio

[0045] Pseudomonas taiwanensis EN-F2 strain (for specific screening process, see CN114369544A), Acinetobacter johnsonii EN-J1 strain (for specific screening process, see CN115011522A), Glutanibacterarilaitensis EM-H8 strain (for specific screening steps, see "Efficient hydroxylamine removal through heterotrophic nitrification by novel bacterium Glutamicibacterarilaitensis EM-H8") and the strains FST-9, FST-12, FST-3 and FST-4 screened in (I) were used to construct a strain composition FST. Specifically, an orthogonal experiment was performed according to Table 2, where 1, 2 and 3 in the table represent 0.1×10 8 CFU / mL, 0.2×10 8 CFU / mL and 0.3×10 8 CFU / mL, and after culturing in nitrification medium at 25 ° C and 150 r / min for 10 h, the removal rates of ammonium nitrogen and COD by microbial agents were determined (Ammonium removal rates represent ammonium nitrogen removal rates, CODremoval rates represent COD removal rates). The results are shown in Table 3. The R value reflects the degree of influence of the inoculation amount of the seven strains on the efficiency of ammonia nitrogen and COD.

[0046] Table 2 Orthogonal experiment

[0047]

[0048] Table 3 Orthogonal experimental test results

[0049]

[0050] As shown in Table 3, the order of influence of Pseudomonas taiwanensis EN-F2, Acinetobacter johnsonii EN-J1, Glutanibacter arilaitensis EM-H8 and the strains FST-9, FST-12, FST-3 and FST-4 screened in (I) on the removal efficiency of ammonium nitrogen is FST-9>FST-4>FST-12>EN-F2>EM-H8>FST-3>EN-J1, and the order of influence on COD removal rate is EN-F2>FST-3>FST-4>EN-J1>FST-12>FST-9>EM-H8. Combined with the optimal inoculum level of single strain (corresponding to the maximum value of k), the best combination of microbial agents for removing ammonium nitrogen is EN-F2:EN-J1:EM-H8:FST-3:FST-4:FST-9:FST-12=3:2:2:1:1:2:1 (agent combination 1), while the combination of microbial agents for removing COD is EN-F2:EN-J1:EM-H8:FST-3:FST-4:FST-9:FST-12=1:3:1:3:2:2:1 (agent combination 2).

[0051] Subsequently, the present application verified the effects of the two combinations, and the results showed that the removal rates of ammonium nitrogen and COD by bacterial agent combination 1 were 77.87% and 50.90%, respectively, and the removal rates of ammonium nitrogen and COD by bacterial agent combination 2 were 80.59% and 70.38%. Based on the removal efficiency of ammonium nitrogen and COD, the optimal ratio of microbial agents was EN-F2:EN-J1:EM-H8:FST-3:FST-4:FST-9:FST-12=1:3:1:3:2:2:1. Under this ratio, the removal rates of ammonium and COD by the microbial agent FST were 10.51 mg / L / h and 277.01 mg / L / h, respectively. The removal rates were significantly higher than those of the microalgae fungal community (the removal effects of ammonium nitrogen and COD were only 0.27 mg / L / h and 2.75 mg / L / h, respectively) and the microbial community isolated and screened from high-salt wastewater (the removal effects of ammonium nitrogen and COD were only 0.27 mg / L / h and 2.75 mg / L / h, respectively).

[0052] (III) Investigate the effects of different culture conditions on the removal of ammonium nitrogen and organic matter by the bacterial agent FST

[0053] In this experiment, glucose was used as the carbon source of the culture medium. The effect of initial temperature on the removal of ammonium nitrogen and organic matter by the optimal bacterial agent FST (i.e., bacterial agent combination 2) screened in (II) was studied through a single factor experiment. The rotation speed was 150r / min and the initial pH value was 8. The removal rate of ammonium nitrogen and organic matter (i.e., COD) by the optimal bacterial agent FST (i.e., bacterial agent combination 2) under the initial temperature of 10℃, 15℃, 20℃, 25℃ and 30℃ was explored (the culture time was 48h). The results are shown in Figure 2. Figure 3 As shown in A.

[0054] Depend on Figure 3 A shows that the bacterial agent FST (i.e., bacterial agent combination 2) has a strong removal ability for both ammonium nitrogen and COD in the range of 15-30°C. When the temperature is 10°C, the OD600 value is the lowest, which is 1.52; the removal rate of ammonium and COD by the bacterial agent FST is only 24.85% and 33.87%, which may be due to the fact that low temperature inhibits the growth of the strain, resulting in its weak ability to remove ammonium and COD (Shenet al., 2020). As the temperature increases, the removal rates of ammonium and COD also continue to rise. When the temperature rises to 25°C, the removal rates of ammonium nitrogen and COD are the highest, at 80.83% and 65.59%, respectively. However, when the temperature rises from 25°C to 30°C, the removal rates of ammonium nitrogen and COD are significantly reduced to 71.94% and 22.88% (p<0.05). Therefore, 25°C was selected as the optimal temperature for the bacterial agent FST to remove ammonium and COD, and subsequent experiments were carried out under this temperature condition.

[0055] In order to further explore the effect of initial rotation speed on the removal of ammonium nitrogen and organic matter by the optimal bacterial agent FST (i.e., bacterial agent combination 2) screened in (II), the initial temperature was 25°C and the initial pH value was 8. The removal rate of ammonium nitrogen and organic matter (i.e., COD) by the optimal bacterial agent FST (i.e., bacterial agent combination 2) under the conditions of initial rotation speed of 0r / min, 50r / min, 100r / min, 150r / min and 200r / min was explored (the culture time was 48h). The results are as follows Figure 3 As shown in B.

[0056] like Figure 3As shown in Figure B, when the speed increased from 0r / min to 100r / min, the removal rate of ammonia nitrogen increased from 39.30% to 53.93%, while the OD600 of the bacterial agent only increased from 1.52 to 1.59. This may be due to the fact that low dissolved oxygen inhibited microbial growth, resulting in a low removal rate of ammonium nitrogen (Hu et al., 2021). When the speed increased to 200r / min, the removal rate of ammonium nitrogen increased significantly to 99.35%, and the OD600 value of the bacterial agent FST increased significantly to 1.77. This result is consistent with previous results that higher dissolved oxygen can significantly improve the removal of inorganic nitrogen by microorganisms (Chen et al., 2022). In addition, when the speed increased from 0r / min to 200r / min, the removal rate of COD also increased significantly from 35.32% to 72.96%. Considering the removal efficiency of ammonium nitrogen and COD, 200r / min was selected as the optimal speed for the bacterial agent to remove ammonium nitrogen and COD, and subsequent experiments were carried out under this speed condition.

[0057] In order to further explore the effect of initial pH on the removal of ammonium nitrogen and organic matter by the optimal bacterial agent FST (i.e., bacterial agent combination 2) screened in (II), the initial temperature was 25°C and the initial speed was 200r / min to explore the removal rate of ammonium nitrogen and organic matter (i.e., COD) by the optimal bacterial agent FST (i.e., bacterial agent combination 2) under pH conditions of 6, 7, 8, 9 and 10 (culture time was 48h). The results are as follows Figure 3 As shown in C.

[0058] like Figure 3 As shown in Figure C, when the initial pH value was 6, the removal rates of ammonia nitrogen and COD were the lowest, at 41.10% and 7.5%, respectively. When the pH values ​​were 9 and 10, the removal rates of ammonia nitrogen were 99.77% and 98.83%, respectively. This phenomenon shows that the bacterial dose FST can still efficiently remove nitrogen under strong alkaline conditions. In addition, increasing the pH value can also significantly increase the COD removal rate. When the pH value is 10.0, the COD removal rate reaches the highest (83.45%). According to previous literature reports, the discharge of strongly acidic or alkaline water will cause serious harm to the environment. According to the National Surface Water Environmental Quality Standards of China, the pH value of surface water should be controlled between 6-9 (Li et al., 2023). In this study, the pH value of the wastewater treated with the bacterial agent FST was maintained between 6.42-7.57. The above results show that the bacterial agent can still play a role in denitrification and carbon removal under alkaline conditions and has the potential to treat actual toilet sewage. Considering the removal characteristics of nitrogen and organic matter by microbial communities and their practical application in toilet sewage, the optimum pH of the bacterial agent FST was determined to be 8.0, and subsequent experiments were carried out under this pH condition.

[0059] (IV) Exploring the nitrification and denitrification characteristics of the microbial agent FST

[0060] In order to explore the denitrification characteristics of the bacterial agent FST (i.e., bacterial agent combination 2), ammonium chloride and sodium nitrate were used as the only nitrogen source. The culture was carried out at 25°C, 200 r / min, and pH 8.0. Samples were taken every 3 hours to measure the ammonium nitrogen, COD, nitrate, nitrite, pH value, and OD in the synthetic wastewater. 600 , the results are as follows Figure 4 As shown in A.

[0061] like Figure 4 As shown in A, after 3 h, the OD of the microbial community 600 The value increased from 1.02 to 1.33, and the removal rates of ammonium nitrogen and COD were only 27.4% and 41.54%. With the growth of the bacterial agent FST, the removal rate of ammonium nitrogen in the first 6 hours was 93.25%, and the OD 600 The maximum ammonium removal rate was 20.2 mg / L / h, which was significantly higher than that of the microbial community FG-06 (7.26 mg / L / h) and the algae-bacteria community 5.87 mg / L / h. During this process, the maximum removal rate of COD can reach 373.78 mg / L / h. However, when the culture time increased from 9 to 12 h, the removal rate of ammonium nitrogen remained unchanged (99%), while the removal rate of COD increased significantly from 65.27% to 76%, indicating that appropriately extending the culture time can improve the removal of COD by the bacterial agent FST.

[0062] To analyze the intermediate and final products of nitrogen transformation during nitrification and denitrification by the bacterial agent FST, single strains were added to the nitrification and denitrification medium at the optimal ratio, and sodium nitrate was used as the nitrogen source. 8 mL of samples were collected in conical flasks at 0 h and 10 h, respectively, and then centrifuged at 6,500 rpm for 10 min. Soluble ammonium nitrogen, nitrite, nitrate, and soluble total nitrogen (TN1) in the supernatant were measured. To measure intracellular nitrogen, 8 mL of culture medium was broken using the Scientz-IID ultrasonic cell disruption system (JY92-IIN, Scientz, Ningbo, China), and then filtered through a 0.22 μm membrane filter (Milli-pore Express PES Membrane, USA). At the same time, ammonium nitrogen, hydroxylamine, nitrate, nitrite, and total nitrogen (TN2) in the supernatant were analyzed. The results are shown in Table 1. Figure 4 As shown in B.

[0063] like Figure 4 As shown in Figure B, in the first 3 hours, the removal rate of nitrate (23.50%) and COD (18.56%) by the inoculum was the lowest, which may be because the bacteria were still in the lag phase. 600The value increased from 1.03 to 1.78, and the removal rates of nitrate and COD reached the highest, 93.97% and 75.25%, respectively. The maximum removal rate of nitrate was 7.47 mg / L / h, which was significantly higher than that of the bacterial agents Mix-CADB A30 (3.10 mg / L / h), Mix-CADA Y23 (0.205 mg / L / h) and Mix-CADA X21 (0.202 mg / L / h). In addition, when removing nitrate, 8.08 mg / L of nitrite could be detected within 3-6 hours, and nitrite was no longer detected after 12 hours. The above results show that the bacterial agent FST can not only carry out heterotrophic nitrification-aerobic denitrification, but also can efficiently remove organic matter.

[0064] (V) Nitrogen balance experiment of bacterial agent FST

[0065] The nitrogen balance experiment was conducted on the bacterial agent FST (i.e., bacterial agent combination 2), and the specific steps were as follows: the bacterial agent FST was added to the nitrification medium in the manner of combination 2, and then 8 ml of samples were collected from each conical flask and centrifuged at 6,500 r / min for 10 min to determine the soluble ammonium nitrogen, nitrite, nitrate, and soluble total nitrogen (TN1) in the supernatant; to determine the intracellular nitrogen content, another 8 ml sample was taken using the Scientz-IID ultrasonic cell disruption system (JY92-IIN, Scientz, Ningbo, China), and then filtered through a 0.22 μm membrane filter (Milli-pore Express PES membrane, USA). At the same time, ammonium, hydroxylamine, nitrate, nitrite, and total nitrogen (TN2) in the supernatant were analyzed. After 10 hours of incubation, two tubes of 8 ml samples were collected from each conical flask, and the above experimental steps were repeated to detect the nitrogen content. According to the literature previously published by Ke et al., the production rates of inorganic nitrogen, intracellular nitrogen, organic nitrogen and gaseous nitrogen in the samples were calculated, and the results are shown in Table 4 (InitialTN represents the initial TN concentration, Final nitrogen concentrations represents the final nitrogen concentration, Intracellular-N represents intracellular nitrogen, and TN removal represents the TN removal rate).

[0066] Table 4 Results of nitrogen balance experiment of bacterial agent FST

[0067]

[0068] As shown in Table 4, when ammonium nitrogen is the only nitrogen source, the initial inorganic nitrogen is mainly converted into nitrogen-containing gas (47.73%) and intracellular nitrogen (41.52%), of which a small amount of inorganic nitrogen is converted into organic nitrogen (12.18±1.52 mg / L) and nitrate (2.72±1.22 mg / L). The bacterial agent can also convert most of the ammonium nitrogen into intracellular nitrogen and nitrogen-containing gas. In addition, during the process of removing ammonium nitrogen by the bacterial agent, the intracellular nitrogen concentration is much higher than that of a single pure strain, which is of great significance for mitigating the impact of the greenhouse effect on the environment. When the bacterial agent FST removes nitrates, the nitrogen produced in the cell can reach 13.25±0.94 mg / L, of which 59.33% of the inorganic nitrogen can be converted into nitrogen-containing gas.

[0069] (VI) Exploring the removal characteristics of high concentrations of ammonium nitrogen and total phosphorus by the bacterial agent FST

[0070] In order to explore the removal characteristics of high-concentration ammonium nitrogen and total phosphorus by the bacterial agent FST (i.e., bacterial agent combination 2), the culture medium was cultured at 25°C, 200r / min and pH 8.0 in a nitrification medium. Samples were taken every 3 hours. The content of ammonium chloride in the nitrification medium was changed, and the ammonium nitrogen concentration was set to 150, 200, 250, 300 and 350 mg / L, respectively. The concentrations of ammonium nitrogen, hydroxylamine, nitrite and nitrate were measured every 12 hours. The ability of the bacterial agent to remove total phosphorus was explored in the phosphorus removal medium. The concentrations of phosphate were set to 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L and 125 mg / L, respectively. The concentrations of total phosphorus and ammonium nitrogen in the culture medium were measured every 24 hours. The results are shown in Figure 2. Figure 5 As shown in A.

[0071] like Figure 5 As shown in A, after 36 h of cultivation, the ammonium nitrogen content decreased from 150, 200, 250 and 300 mg / L to 0.32, 0.26, 0.35 and 0.48 mg / L, respectively, and the corresponding ammonium nitrogen removal rates were 99.79%, 99.87%, 99.86% and 99.84%, respectively. However, when the culture time was extended to 48 h, the ammonium nitrogen content increased, which may be due to bacterial death (Ouyang et al., 2020). When the initial concentration of ammonium nitrogen was 350 mg / L, the bacterial agent FST could remove 96.18% of ammonium nitrogen within 48 h. Only a small amount of nitrification intermediates (NO3-N <10 mg / L) were detected throughout the ammonium oxidation process, and its short-range denitrification pathway can shorten the time for bacterial agents to treat wastewater.

[0072] To further explore whether FST (i.e., bacterial agent combination 2) can remove high concentrations of total phosphorus, the inoculation amount of FST (i.e., bacterial agent combination 2) was 1.2×10 8CFU / mL was inoculated into simulated wastewater with total phosphorus contents of 25, 50, 75, 100 and 125 mg / L (containing 0.573 g / L ammonium chloride, 7.69 g / L sodium acetate, 0.04 g / L magnesium sulfate, 0.009 g / L iron sulfate, 1.5 g / L potassium dihydrogen phosphate and 0.014 g / L calcium chloride) and cultured. Samples were taken once every 24 hours to test the total phosphorus concentration in the system. The results are as follows Figure 5 As shown in B.

[0073] like Figure 5 As shown in Figure B, after 24 hours of cultivation, the removal rate of the bacterial agent for the initial total phosphorus concentration of 25 mg / L can reach 1.00 mg / L / h (data not shown). In addition, when TP is 50 mg / L, the TP removal efficiency drops significantly to 40.47%, which is lower than the aerobic microbial agent (71.09%) isolated from activated sludge. When the TP concentration is higher than 50 mg / L, the total phosphorus removal rate is lower than 33%. This may be because the nitrogen and carbon source content in the culture medium decreases after a period of cultivation, which cannot meet the needs of bacterial growth and reproduction, resulting in a decrease in the phosphorus absorption rate of the bacterial agent FST.

[0074] (VII) Explore the ability of the bacterial agent FST to remove nitrogen and phosphorus from actual toilet sewage

[0075] In order to explore the ability of bacterial agent FST (i.e., bacterial agent combination 2) to remove nitrogen and phosphorus from actual toilet sewage, the present application conducted the following experiments:

[0076] Toilet sewage was collected from Qianxi County, Guizhou Province, and four treatment groups (C1, C2, C3, and C4) were set up, where C1 represented toilet sewage, and C2 represented toilet sewage inoculated with bacterial agent FST (i.e., bacterial agent combination 2) (inoculation amount was 1.2×10 8 CFU / mL), C3 represents toilet sewage with carbon source added (addition amount is 45.66 g / L), and C4 represents toilet sewage with carbon source added (addition amount is 45.66 g / L) and inoculated with bacterial agent FST (inoculation amount is 1.2×10 8 CFU / mL). All samples were cultured at 25°C, 200 rpm and pH 8.0 for 7 days. After 1 day, 3 days, 5 days and 7 days, the concentrations of ammonia nitrogen, nitrate, total nitrogen and total phosphorus in each group were tested. The results are as follows: Figure 6 shown.

[0077] like Figure 6As shown, after 7 days of cultivation, the ammonium nitrogen efficiency in the four treatment groups was decreasing (6A), among which the ammonium nitrogen removal rate in the toilet sewage experimental group was 44.14%, the ammonium nitrogen removal rate in the experimental group with only the bacterial agent FST was 36.03%, the ammonium nitrogen removal rate in the experimental group with only the carbon source was 60.98%, and the ammonium nitrogen removal rate in the experimental group with both the bacterial agent and the carbon source was the highest, at 72.55%. In addition, on the 5th day, it was observed that the ammonium nitrogen content in the experimental group with only the bacterial agent FST was higher than that in the toilet sewage treatment, and the OD of the bacteria was 600 The value is also decreasing (data not shown), which may be due to the lack of carbon source in the late stage of cultivation, which leads to bacterial death. The above results also further show that adding carbon source and bacterial agent to toilet sewage can significantly improve the removal rate of ammonium nitrogen in sewage, and the maximum removal rate of ammonium nitrogen can reach 6.55mg / L / h, which is much higher than the microbial agent obtained from shrimp farm wastewater (5.22mg / L / h) and the isolated strain Pseudomonas fluorescens2P24 (1.89mg / L / h). While the ammonium nitrogen content is decreasing, the total nitrogen content is also decreasing. Compared with the two experimental groups where only bacterial agent and carbon source are added to toilet sewage, the experimental group where both are added has a faster total nitrogen removal efficiency, which can reach 58.99% (6C), which is 11.37% higher than the toilet sewage experimental group before strengthening. At the same time, after adding bacterial agent and carbon source to toilet sewage, the total phosphorus removal efficiency can reach 64.25% (6D), which is significantly higher than the experimental group without adding bacterial agent and carbon source (51.57%). The above experimental results show that the combined addition of bacterial agent FST and carbon source can significantly promote the removal of nitrogen and phosphorus in toilet sewage.

[0078] In summary, this application combined the four highly efficient denitrification and carbon removal bacteria isolated and screened from actual toilet sewage with the highly efficient denitrification and denitrification bacteria obtained in the laboratory in the early stage to construct the microbial agent FST, which has the ability to efficiently degrade NH4 + -N, NO3 - -N and COD. The nitrogen balance results showed that 47.73% of the initial ammonium nitrogen could be converted into nitrogen-containing gas through heterotrophic nitrification, and no accumulation of intermediate nitrite was detected during the removal of ammonium nitrogen by the agent; when the agent FST performed aerobic nitrification, the conversion rate of gaseous nitrogen could reach 59.33%. In addition, after adding the microbial agent FST to the actual unsterilized toilet sewage, the removal rates of ammonium nitrogen, total nitrogen and total phosphorus increased by 28.14%, 11.37% and 12.68% respectively.

[0079] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A strain composition, characterized in that: include Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providence huaxiensis FST-4 strain, Testosterone communion FST-9 strain and Proteus foods FST-12 strain, described Pseudomonas Taiwanese The deposit number of EN-F2 strain is CCTCC NO: M 20211515. Acinetobacter johnsonii The deposit number of EN-J1 strain is CCTCC NO: M2022702. Glutanibacter arilaitensis The deposit number of EM-H8 strain is CCTCC NO: M 20241974. Leucobacter denitrifying The deposit number of FST-3 strain is CCTCC NO: M 20241977. Providence huaxiensis The deposit number of FST-4 strain is CCTCC NO: M 20241978. Comamonas Testosterones The deposit number of FST-9 strain is CCTCC NO: M 20241975. Proteus foods The deposit number of the FST-12 strain is CCTCC NO: M 20241976.

2. The strain composition according to claim 1, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providence huaxiensis FST-4 strain, Testosterone communion FST-9 strain and Proteus foods The colony ratio of the FST-12 strain is 0.5-3.5:1.5-3.5:0.5-2.5:0.5-3.5:0.5-2.5:1.5-2.5:0.5-1.

5.

3. The strain composition according to claim 2, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providence huaxiensis FST-4 strain, Testosterone communion FST-9 strain and Proteus foods The colony ratio of the FST-12 strain was 1-3:2-3:1-2:1-3:1-2:1.5-2.5:0.5-1.

5.

4. The strain composition according to claim 2, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providence huaxiensis FST-4 strain, Testosterone communion FST-9 strain and Proteus foods The colony ratio of the FST-12 strain is 2.5-3.5:1.5-2.5:1.5-2.5:0.5-1.5:0.5-1.5:1.5-2.5:0.5-1.

5.

5. The strain composition according to claim 4, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providence huaxiensis FST-4 strain, Testosterone communion FST-9 strain and Proteus foods The colony ratio of the FST-12 strain was 3:2:2:1:1:2:

1.

6. The strain composition according to claim 2, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arylamide EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi The colony ratio of the FST-12 strain is 0.5-1.5:2.5-3.5:0.5-1.5:2.5-3.5:1.5-2.5:1.5-2.5:0.5-1.

5.

7. The strain composition according to claim 6, characterized in that Said Pseudomonas taiwanensis EN-F2 strain, Acinetobacter johnsonii EN-J1 strain, Glutanibacter arilaitensis EM-H8 strain, Leucobacter denitrificans FST-3 strain, Providencia huaxiensis FST-4 strain, Comamonas testosteroni FST-9 strain and Proteus cibi The colony ratio of the FST-12 strain was 1:3:1:3:2:2:

1.

8. Use of the strain composition according to any one of claims 1 to 7 in treating toilet sewage.

9. A toilet sewage treatment agent, characterized in that: The invention comprises the strain composition according to any one of claims 1 to 7.

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