A composite microbial flora, a microbial agent and application thereof

Abstract

The application discloses a kind of microbial complex bacterial population, microbial inoculum and application thereof, and is related to microorganism and application technical field.The microbial complex bacterial population includes Acinetobacter sichuanensis LHH12, Pseudomonas songnenensis SJ2-76, Diaphorobacter nitroreducens SYY4 and Thauera aminoaromatica SJ2-12.The microorganism for constructing complex bacterial population is obtained from semi-coke wastewater, and has good phenol tolerance and chemical oxygen demand (COD) degradation capacity, and can be used for purifying high-phenol wastewater, biological denitrification and phosphorus removal and degrading COD.The microbial complex bacterial population of the application can effectively improve the COD degradation rate in phenol ammonia wastewater and effectively reduce the treatment cost of high-phenol ammonia wastewater such as semi-coke without changing the original microbial community composition of wastewater through the synergistic effect between strains, which has great potential for promoting low-carbon green and sustainable development of coal chemical industry.

Description

Technical Field

[0001] This invention relates to the field of microbiology and its application technology, specifically to a microbial complex, microbial agent and its application. Background Technology

[0002] Coal, as a vital energy resource in my country, plays a crucial role in the national economic development. For example, semi-coke, produced from coal through low-temperature (450-650℃) dry distillation, is widely used in industry due to its low pollution and low emissions. Semi-coke is an indispensable carbon material in the global energy transition, with an annual production exceeding 100 million tons. However, its production not only consumes large amounts of water resources but also generates significant amounts of phenol and ammonia wastewater. It is reported that my country generates approximately 30 million cubic meters of semi-coke wastewater annually. 3 These wastewaters are complex in composition, containing not only inorganic pollutants such as cyanide, ammonia nitrogen, thiocyanate, and heavy metals, but also recalcitrant organic pollutants such as phenols, benzene compounds, polycyclic aromatic hydrocarbons, and heterocyclic compounds containing nitrogen, oxygen, and sulfur. They also contain a large amount of toxic substances that inhibit the growth of microorganisms, with ammonia nitrogen reaching as high as 5000 mg / L, phenols exceeding 5000 mg / L, and chemical oxygen demand (COD) reaching 30000-40000 mg / L. They are characterized by high color, high toxicity, and poor biodegradability, and are a typical type of highly polluted and recalcitrant industrial wastewater. Direct discharge would pose a great threat to human health and the living environment, and must be treated before discharge or reuse.

[0003] Semi-coke wastewater is mostly grayish-black, and the main pollutants are ammonia nitrogen and phenols. Since its composition is similar to that of coking wastewater, its treatment technology mainly draws on the treatment process of coking wastewater. However, semi-coke wastewater contains a large number of low- and medium-molecular-weight pollutants that have not been oxidized at high temperatures. Its composition is more complex than that of coking wastewater, and the pollutant concentration and COD content are about 10 times higher than those in coking wastewater. Therefore, the treatment of semi-coke wastewater requires different treatment technologies.

[0004] Currently, the main treatment technology for semi-coke wastewater adopts a three-stage treatment process of "pretreatment + biochemical treatment + advanced treatment". Among them, biochemical treatment, which utilizes microorganisms to degrade pollutants through metabolism, is an important part of wastewater treatment. However, due to the inhibitory effect of high concentrations of phenols and other substances on bacterial activity, the biochemical treatment effect of semi-coke wastewater is limited. Summary of the Invention

[0005] This invention addresses the problem of unsatisfactory biochemical treatment effects of semi-coke wastewater by providing a microbial composite flora, bacterial agent, and its application. It involves isolating and screening highly effective bacterial strains with COD degradation capabilities and phenol tolerance from semi-coke wastewater. Simultaneously, drawing inspiration from the activated sludge process in wastewater treatment, it utilizes highly efficient flocculating bacterial strains isolated from semi-coke wastewater to form a composite microbial flora. Without altering the original microbial community composition of the semi-coke phenol-ammonia wastewater, the synergistic effect among the various strains effectively improves the COD degradation rate and enhances biological nitrogen and phosphorus removal. This has significant potential in reducing wastewater treatment costs and achieving wastewater purification and compliance with discharge standards.

[0006] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, this invention discloses a complex microbial community, including Acinetobacter chuanxiensis (… Acinetobacter sichuanensis ), Songnen Plain Pseudomonas ( Pseudomonas songnenensis )and Diaphorobacter nitroreducens .

[0008] In a preferred embodiment of the present invention, the Acinetobacter chuanxiensis ( Acinetobacter sichuanensis Acinetobacter Sichuanus ( Acinetobacter sichuanensis LHH12 was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232665.

[0009] In a preferred embodiment of the present invention, the *Pseudomonas spp.* (Songnen Plain) Pseudomonas songnenensis ) is Pseudomonas stolonifera of Songnen Plain ( Pseudomonas songnenensis SJ2-76 was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232667.

[0010] In a preferred embodiment of the present invention, the Diaphorobacter nitroreducens for Diaphorobacter nitroreducens SYY4 was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232669.

[0011] In a preferred embodiment of the present invention, it also includes *Dauerella aromatica* (a type of fungus). Thauera aminoaromatica SJ2-12 was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232668.

[0012] In a second aspect, the present invention discloses a composite microbial agent comprising the composite microbial community as described in the first aspect.

[0013] In a preferred embodiment of the present invention, the Acinetobacter chuanxiensis ( Acinetobacter sichuanensis LHH12, Pseudomonas spp. of Songnen Plain ( Pseudomonas songnenensis SJ2-76, Aromatic Amino Acids Tauella ( Thauera aminoaromatica SJ2-12 and Diaphorobacter nitroreducens The effective viable count ratio of SYY4 is 1:1:(0.6-0.8):(0.6-0.8).

[0014] Thirdly, the present invention discloses the application of a composite microbial community as described in the first aspect or a composite microbial agent as described in the second aspect in the purification of high-phenol wastewater and biological nitrogen and phosphorus removal, wherein the phenol concentration in the wastewater is ≥100 mg / L.

[0015] Fourthly, this invention discloses the application of a composite microbial community as described in the first aspect or a composite microbial agent as described in the second aspect in the degradation of chemical oxygen demand (COD) in wastewater.

[0016] Preferably, the inoculation amount of the composite microbial flora or composite microbial agent is 0.8-1.2%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention utilizes strains isolated from semi-coke wastewater that possess resistance to phenol and COD degradation, as well as the ability to form flocs, to construct a microbial complex. In semi-coke wastewater with an equal volume of inorganic salt culture medium, the COD removal rate can reach 68%. The microbial complex provided by this invention draws inspiration from the activated sludge process in wastewater treatment. Without altering the original microbial community structure in high-phenol and ammonia wastewater such as semi-coke, it effectively enhances the COD degradation capacity of the wastewater through the synergistic effect of various strains, while simultaneously strengthening biological nitrogen and phosphorus removal. This has great potential in reducing wastewater treatment costs and achieving wastewater purification and compliance with discharge standards. Attached Figure Description

[0019] Figure 1 This is a diagram showing the main microbial composition (genus level) in semi-coke phenol-ammonia wastewater samples. SJ is a semi-coke wastewater sample collected from the hydrolysis tank; H is a semi-coke wastewater sample collected from the anaerobic fluidized bed device.

[0020] Figure 2 This is a graph evaluating the phenol tolerance of the isolated strains;

[0021] Figure 3This is a schematic diagram of the COD degradation in phenol-ammonia wastewater by isolated strains. In the diagram, wastewater in a comes from Yulin, Shaanxi, and wastewater in b comes from Naomuhu, Xinjiang.

[0022] Figure 4 These are morphological images of various strains in a microbial complex. A shows the morphology of the strains when they are stationary; B shows the morphology of the strains when they are shaken during culture; and C shows the optical microscopy staining images of each strain. The scale bar is 1 μm.

[0023] Figure 5 This is a schematic diagram illustrating the degradation of COD in phenol-ammonia wastewater by a complex microbial community.

[0024] Figure 6 It is an aromatic amino acid-producing bacterium (Dauerella) Thauera aminoaromatica A diagram of denitrification-related genes present in the SJ2-12 genome. Detailed Implementation

[0025] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents or materials used in the following examples are all from commercial sources.

[0026] The culture medium used in the embodiments of this invention is as follows:

[0027] The formula for R2A liquid culture medium (1L) is as follows: yeast extract 0.5 g, tryptone 0.5 g, magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, dipotassium hydrogen phosphate 0.3 g, starch 0.5 g, glucose 0.5 g (filtered for sterilization), enzymatically hydrolyzed casein 0.5 g, pH adjusted to 7.0±0.2;

[0028] The formula for R2A solid medium (1L) is as follows: yeast extract 0.5 g, tryptone 0.5 g, magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, dipotassium hydrogen phosphate 0.3 g, starch 0.5 g, glucose 0.5 g (filtered for sterilization), enzymatically hydrolyzed casein 0.5 g, agar 15 g, and pH adjusted to 7.0±0.2.

[0029] The formulation of LB liquid medium (1L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, adjusted to pH 7.0±0.2;

[0030] The formula for LB solid medium (1L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, adjust pH to 7.0±0.2;

[0031] The formulation of inorganic salt (MSM) basal medium (1L): Na₂HPO₄ 2800 mg, (NH₄)₂SO₄ 500 mg, CuCl₂·2H₂O 0.001 mg, H₃BO₃ 0.03 mg, FeSO₄·7H₂O 0.2 mg, MnCl₂·4H₂O 0.003 mg, NiCl₂·6H₂O 0.02 mg, KH₂PO₄ 1000 mg, Na₂EDTA 0.5 mg, CoCl₂ 2· Add 0.02 mg of 6H2O, 0.01 mg of ZnSO4·7H2O, and 0.003 mg of Na2MoO4·2H2O to adjust the pH to 7.2±0.1.

[0032] Formula for phenol-resistant solid medium (1L): 1L MSM basal medium + different concentrations of phenol, 15 g agar, adjust pH to 7.0±0.2;

[0033] In this embodiment, four microorganisms used to construct the composite microbial community were deposited at the China Center for Type Culture Collection (CCCGC) for patented strain preservation. The specific address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. After strain verification and activity testing, the CCCGC issued a patent preservation certificate on December 25, 2023. Among them, Acinetobacter chuanxiensis (… Acinetobacter sichuanensis The preservation number of LHH12 is CCTCC M 20232665; *Pseudomonas s. Songnenpuria* ( Pseudomonas songnenensis The accession number for SJ2-76 is CCTCC M 20232667; Aromatic amino acid Dauerella ( Thauera aminoaromatica The accession number for SJ2-12 is CCTCC M 20232668; Diaphorobacter nitroreducens The accession number for SYY4 is CCTCC M 20232669.

[0034] The following is a description through specific embodiments.

[0035] Example 1: Analysis of Microbial Community Structure in Semi-coke Wastewater

[0036] In this embodiment, the semi-coke wastewater used was collected from the hydrolysis tank of Shaanxi Wanbangda Water Co., Ltd. and the anaerobic fluidized bed device of United Filtration Technology (Wuhan) Co., Ltd. After high-speed centrifugation, the collected semi-coke wastewater samples were used to extract the total genome from the centrifuged precipitate using an environmental genomics extraction kit (Omega). The extracted environmental genomics samples were then sent to a sequencing company for 16S rRNA gene amplicon high-throughput sequencing to analyze the microbial community structure and composition in the semi-coke wastewater.

[0037] The top 20 microbial community compositions (at the genus level) in the two sample groups (SJ is the sample from the hydrolysis tank of Shaanxi Wanbangda Water Co., Ltd., and H is the sample from the anaerobic fluidized bed device of United Filtration Technology (Wuhan) Co., Ltd.) are as follows: Figure 1 As shown, the main microorganisms in the hydrolysis tank (SJ) are mainly aerobic or facultative anaerobic bacteria. Figure 1 a), among which, the most abundant is the competitive bacterium (Candidatus). -Competibacter Thiobacillus spp. Thiobacillus ) and Cyclophila spp. ( Alicycliphilus ), followed by Ottobacter spp. ( Ottowia ), Prolongomonas spp. Extensimonas ), Taureella genus ( Thauera The microbial composition of this fluidized bed exhibits a clear dominance of specific bacterial genera. In contrast, the microorganisms in the anaerobic fluidized bed (H) are predominantly strict anaerobic bacteria. Figure 1 b), among which, strictly anaerobic Clostridium species ( Clostridium The relative abundance of ) was the highest, followed by Ottobacter spp. ( Ottowia Thiobacillus ( ) Thiobacillus ), Clostridium erythropoiesis Erysipelatoclostridium ), Methanobacterium bristlenoides ( Methanosaeta The distribution of its fungal genera is relatively more uniform.

[0038] The dominant bacterial genera in both groups of samples included Thiobacillus ( ). Thiobacillus ), genus Cyclophila ( Alicycliphilus Ottosporum spp. Ottowia ) and Tauella ( Thauera This indicates that these genera are relatively dominant in the treatment of high-phenol and ammonia wastewater such as semi-coke.

[0039] Example 2 Isolation and Identification of Microbial Strains

[0040] (1) Isolation of microorganisms:

[0041] The semi-coke wastewater used for microbial isolation and screening originated from the hydrolysis tank of Shaanxi Wanbangda Water Co., Ltd. and the anaerobic fluidized bed device of United Filtration Technology (Wuhan) Co., Ltd. The collected semi-coke wastewater samples were serially diluted with sterile water (10⁻⁶ ppm). -l 10 -2 10 -3 10 -4 10 -5The diluted solution was then spread onto solid plates such as R2A and LB, and the plates were incubated at 28°C for 2-3 days. After the bacteria grew on the plates, a single colony was picked and inoculated into R2A or LB liquid medium and cultured with shaking (28°C, 180 rpm / min). The bacterial culture grown in the liquid medium was then inoculated onto the corresponding new solid plates for streaking purification to obtain a pure culture of the bacterial strain. The pure culture was stored in 40% glycerol (1:1 by volume) and kept at -80°C for later use.

[0042] (2) Identification of microorganisms:

[0043] The isolated strains were amplified by PCR using universal primers for the 16S rRNA gene (27F and 1492R, primer sequences 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The amplified products were sent to a biotechnology company for sequencing. The sequencing results were compared with BLAST sequences in NCBI to obtain the taxonomic unit to which the isolated strains belonged.

[0044] We compared the results of the isolation and identification of the strains with the results of the high-throughput analysis of the microbial community structure in semi-coke wastewater in Example 1. We found that the multiple genera of bacteria isolated and identified from the semi-coke wastewater, as shown in Table 1, included dominant genera in the wastewater, such as *Lipidobacterium*. Alicycliphilus ), Taureella spp. Thauera Ottosporum spp. Ottowia ), Pseudomonas ( Pseudomonas This includes other genera such as Acinetobacter ( ) and others. Acinetobacter ), Paracoccus ( Paracoccus Thermomonas spp. Thermomonas ), and beneficial bacteria ( Diaphorobacter ) and Agaricomyces ( Zoogloea These genera, such as [list of bacteria], were also confirmed in the high-throughput results, and are all part of the microbial community composition in semi-coke wastewater.

[0045] Table 1. Taxonomic units to which the isolated strains belong

[0046] strain number Genus to which the strain belongs strain number Genus to which the strain belongs SJ2-3 SJ2-16 SJ2-2 SJ2-12 SXYN20 SJ2-80 JY23-2 LHH12 SYY4 SJ2-76 SJ2-30-2

[0047] Example 3 Evaluation of the phenol tolerance of the isolated strain

[0048] To evaluate the tolerance of the isolated strains (as shown in Table 1) to phenol, the growth of the isolated strains was observed using MSM basal medium with phenol as the sole carbon source. First, MSM solid medium containing different concentrations of phenol (100, 200, 500, 1000, 1500 mg / L) was prepared. Then, single colonies obtained by streaking were streaked at -80℃ and spotted onto different plates, with R2A plates used as a positive control.

[0049] The results are as follows Figure 2 As shown, none of the strains could grow on plates with a phenol concentration of 1500 mg / L. (Acinetobacter chuanxiensis) Acinetobacter sichuanensis LHH12 can grow on plates with phenol concentrations of 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L; *Pseudomonas spp.* (Songnen Plain) Pseudomonas songnenensis SJ2-76 can grow on plates with phenol concentrations of 100 mg / L, 200 mg / L, and 500 mg / L, but cannot grow on plates with a phenol concentration of 1000 mg / L. Diaphorobacter nitroreducens SYY4 and Paracoccus variolidiniae ( Paracoccus versutus SJ2-80 can grow on plates with a phenol concentration of 100 mg / L, but cannot grow on plates with phenol concentrations of 200 mg / L, 500 mg / L, or 1000 mg / L. Other strains also cannot grow on plates with a phenol concentration of 100 mg / L. This indicates that *Acinetobacter chuanxiensis* (SJ2-80) is not a viable growth factor. Acinetobacter sichuanensis LHH12 showed the strongest resistance to phenol, followed by Pseudomonas spp. (Songnen Plain) Pseudomonas songnenensis )SJ2-76. Diaphorobacter nitroreducens SYY4 and Paracoccus variolidiniae ( Paracoccus versutus SJ2-80 has a certain resistance to phenol.

[0050] Example 4 Evaluation of the degradation capacity of isolated strains of chemical oxygen demand (COD) in high-phenol and ammonia wastewater such as semi-coke.

[0051] The main evaluation focused on the degradation capabilities of phenol-tolerant strains LHH12, SJ2-76, SYY4, and SJ2-8 in Example 3 for chemical oxygen demand (COD). Previous studies have reported that *Daucus* spp. (…) Thauera This strain not only possesses denitrification capabilities but can also degrade various phenolic compounds, making it valuable for application in petrochemical wastewater treatment, especially in high-phenolic wastewater. Therefore, this embodiment evaluates the COD degradation capabilities of the aforementioned phenol-resistant strains, including strains LHH12, SJ2-76, SYY4, and SJ2-80, while also assessing the COD degradation capabilities of the isolated aromatic amino acid-producing *Daucus* strain. Thauera aminoaromaticaThe COD degradation capacity of SJ2-12 was evaluated.

[0052] The semi-coke wastewater used in this experiment came from the semi-coke wastewater collection pond of Shaanxi Yulin Wanbangda Water Co., Ltd., and from Naomohu Lake in Xinjiang, respectively. This embodiment used semi-coke wastewater collected from Yulin, Shaanxi, and Naomohu Lake in Xinjiang as culture media (wherein, an equal volume of MSM inorganic salt basal medium was added to the semi-coke wastewater collected from Naomohu Lake in Xinjiang) to evaluate the COD degradation capacity of the above-mentioned strains. The specific implementation method is as follows:

[0053] First, the bacterial strains preserved at -80℃ were streaked onto R2A solid medium. Single colonies were picked from the plates and inoculated into glass tubes containing 3 mL of R2A liquid medium. The tubes were then incubated at 28℃ and 180 rpm for 24-36 hours. The OD values ​​of the different bacterial cultures were then measured. 600 Value (when OD) 600 When the value is between 0.6 and 0.8, it indicates that the bacteria are in the vigorous logarithmic growth phase. Then, the logarithmic growth phase bacterial solution was inoculated at a 1% inoculum into sterilized and cooled to room temperature semi-coke wastewater culture medium (without any added nutrients). At the same time, semi-coke wastewater culture medium without added bacterial solution was used as a blank control. The culture was carried out in a shaker at 28℃ and 180 rpm / min, and the COD change in the wastewater was sampled and detected at regular intervals.

[0054] The determination of chemical oxygen demand (COD) follows the rapid digestion spectrophotometric method specified in HJ / T 399-2007, "Environmental Protection Industry Standard of the People's Republic of China". The detailed steps are as follows:

[0055] (1) Take 1 mL of the wastewater to be tested from the experimental group and the control group respectively, dilute it 3 times with double-distilled water, and centrifuge the diluted sample at 12,000 rpm for 5 min (the culture medium made from the semi-coke wastewater collected in Xinjiang does not need to be diluted when testing, just take 3 mL of the wastewater to be tested and centrifuge it directly).

[0056] (2) Take 2 mL of the supernatant after centrifugation and mix it with 5 mL of high-concentration pre-prepared reagent (i.e., 665 μL potassium dichromate solution + 335 μL mercuric sulfate solution + 4 mL silver sulfate-sulfuric acid solution). The blank background solution is prepared by mixing 2 mL of distilled water with 5 mL of high-concentration pre-prepared reagent.

[0057] (3) Place the mixed sample into a digester and digest it at 165°C for 15 min, then allow it to cool naturally to room temperature.

[0058] (4) Using the blank background solution as a reference solution, adjust the zero point and measure the absorbance of the solution sequentially at a wavelength of 600 nm;

[0059] (5) Calculate the COD value of the sample to be tested based on the standard curve.

[0060] The results are as follows Figure 3 As shown, the semi-coke wastewater collected in Yulin, Shaanxi Province ( Figure 3 a) Without adding any nutrients, Acinetobacter Sichuanis ( Acinetobacter sichuanensis In the LHH12 culture medium, the COD content decreased to below 3050 mg / L within 25 minutes, and remained below 3100 mg / L within 50 minutes. Although the COD content increased slightly between 50 and 75 minutes, it still remained below 3250 mg / L, and continued to decrease over the subsequent 75-100 minutes, indicating that Acinetobacter chuanxiensis (…) Acinetobacter sichuanensis LHH12 has a good and stable COD degradation effect.

[0061] Adding Pseudomonas sineuri (Songnen Plain Pseudomonas) Pseudomonas songnenensis In the semi-coke wastewater of SJ2-76, the COD content dropped to below 3150 mg / L within 50 minutes, and remained stable at around 3150 mg / L within 100 minutes, indicating that it has a good COD degradation effect.

[0062] In addition, among the above five strains, *Paragonimus westermani* (…) Paracoccus versutus SJ2-80 and Aromatic Amino Acids Tauella ( Thauera aminoaromatica SJ2-12 also exhibits a certain COD degradation effect, reducing the COD content to below 3400 mg / L within 25 minutes. Diaphorobacter nitroreducens SYY4 has a relatively weak COD removal capacity.

[0063] Regarding the semi-coke wastewater collected from Naomohu Lake in Xinjiang ( Figure 3 b) In medium supplemented with an equal volume of MSM inorganic salts, Diaphorobacter nitroreducens SYY4 exhibits the strongest COD degradation capability, reducing COD levels to below 450 mg / L within 75 minutes and even below 350 mg / L within 100 minutes. Furthermore, the COD level remains stable below 400 mg / L for the next 20 minutes.

[0064] Acinetobacter chuanxiensis ( Acinetobacter sichuanensis LHH12 showed a stable decreasing trend within 120 minutes, with the COD content steadily decreasing from 1050 mg / L to below 700 mg / L, demonstrating good COD degradation ability.

[0065] In addition, Pseudomonas aeruginosa of Songnen Plain (Pseudomonas songnenensis SJ2-76 also has a certain COD degradation effect, while Paracoccus variegata (SJ2-76) Paracoccus versutus SJ2-80 and Aromatic Amino Acids Tauella ( Thauera aminoaromatica SJ2-12 has the weakest COD removal capacity.

[0066] Example 5: Evaluation of the ability of microbial complex communities to degrade chemical oxygen demand (COD) in high-phenol and ammonia wastewater such as semi-coke.

[0067] This embodiment observed the morphology of the above-mentioned strains LHH12, SJ2-76, SYY4, SJ2-80, and SJ2-12, such as... Figure 4 As shown, Diaphorobacter nitroreducens SYY4 and Aromatic Amino Acids (Dauerella) Thauera aminoaromatica SJ2-12 forms stable bioflocs during its growth. Figure 4 A), the bioflocs formed when at rest sink to the bottom of the test tube under the influence of gravity. Figure 4 B), the supernatant is clear and transparent. Under a microscope, the bacterial cells are clustered together and have a relatively compact structure. Figure 4 C), can be used to attach to other microorganisms; Pseudomonas sinepec (Songnen Plain Pseudomonas) Pseudomonas songnenensis SJ2-76 also forms a membrane structure during its growth process. Figure 4 A and 4B), but their structures are unstable, and the supernatant is relatively turbid; while Acinetobacter chuanxiensis (A and 4B) Acinetobacter sichuanensis LHH12 and Paracoccus variegata ( Paracoccus versutus SJ2-80 remains uniformly turbid throughout its growth process and grows relatively quickly. Under a microscope, its cells are found to be dispersed.

[0068] Based on the above experimental results, this embodiment selected three strains with good COD degradation effect on semi-coke wastewater, namely Acinetobacter chuanxiensis (… Acinetobacter sichuanensis LHH12 Diaphorobacter nitroreducens SYY4 and Songnen Plain Pseudomonas ( Pseudomonas songnenensis SJ2-76 was selected as a potential strain for constructing a complex microbial community.

[0069] Meanwhile, this embodiment draws on activated sludge from municipal wastewater treatment and also selects *Daucus* genus, one of the dominant bacterial groups in wastewater treatment systems. Thauera ) strain, namely Aromatic Amino Acids Tauerella ( T. aminoaromatica SJ2-12 (this strain can not only form bioflocs, but also has a certain COD removal effect) is used as a potential strain to improve the denitrification and phosphorus removal effect of the microbial complex.

[0070] First, this embodiment used the streak-line method to conduct an antagonistic experiment on the above four strains, and the results showed that there was no mutual inhibition among these four strains. Second, this invention used semi-coke wastewater collected from Xinjiang to evaluate the COD degradation capacity of the microbial complex composed of the above four strains. The specific experimental steps are as follows:

[0071] In this embodiment, an equal volume of MSM inorganic salt culture medium was added to semi-coke wastewater collected in Xinjiang as the phenol-ammonia wastewater used in the experiment. The experiment included a blank control group and an experimental group, namely, the blank control group: phenol-ammonia wastewater + distilled water; the experimental group: phenol-ammonia wastewater + compound microbial community. The compound microbial community in the experimental group was divided into three different combinations: combination 1 consisted of SJ2-76 and LHH12, with an effective viable count ratio of 1:1; combination 2 consisted of SJ2-76, LHH12 and SYY4, with an effective viable count ratio of 1:1:(0.6-0.8); combination 3 consisted of SJ2-76, LHH12, SYY4 and SJ2-12, with an effective viable count ratio of 1:1:(0.6-0.8):(0.6-0.8); the mixed bacterial solution (OD) 600 (The value is approximately 0.6) Inoculate the above wastewater at a rate of 1%, and take samples regularly to detect changes in COD in the wastewater.

[0072] like Figure 5 As shown, two strains of bacteria (i.e., [the bacteria]) exhibited good degradation effects in the semi-coke wastewater of Yulin. Figure 5 The bacterial community consisting of two strains (SJ2-76 and LHH12) reduced the COD content in Xinjiang semi-coke wastewater from 1125 mg / L to 700 mg / L when an equal volume of MSM medium was added, resulting in a COD removal rate of approximately 38%. When strain SYY4 (i.e., [missing information]) was added to the above two bacterial communities... Figure 5 The COD content in Xinjiang semi-coke wastewater decreased from 1100 mg / L to 350 mg / L, with a COD removal rate of approximately 68% when three strains of bacteria (SYY4 and SJ2-12) were added to the bacterial community simultaneously. Figure 5 Four strains of bacteria (SJ2-12 and SYY4) were found to reduce the COD content in Xinjiang semi-coke wastewater to below 350 mg / L, a greater reduction than that of the three strains, with a COD removal rate of approximately 68%. Furthermore, while both strain SJ2-12 and SYY4 can form bioflocs during growth, strain SJ2-12 can also grow under anaerobic conditions, producing more stable bioflocs. Moreover, strain SJ2-12 is a dominant strain in activated sludge, possessing denitrification-related genes in its genome, such as... Figure 6 As shown, strain SJ2-12 can therefore be used for biological denitrification treatment.

[0073] In summary, this invention provides a method using Acinetobacter chuanxiensis (… Acinetobacter sichuanensis LHH12 and Songnen Plain Pseudomonas ( Pseudomonas songnenensis SJ2-76, Aromatic Amino Acids Tauella ( Thauera aminoaromatica SJ2-12 Diaphorobacter nitroreducens A microbial complex composed of SYY4. This microbial complex not only has a degradation effect on high-COD semi-coke wastewater collected in Yulin, but also has a removal effect on low-COD semi-coke wastewater collected in Xinjiang.

[0074] This invention draws inspiration from the activated sludge process in municipal wastewater treatment. It utilizes strains isolated from semi-coke wastewater, possessing phenol and COD degradation capabilities and floc-forming abilities, to construct a microbial complex. In semi-coke wastewater with the addition of an equal volume of inorganic salts, the COD removal rate of the complex can reach 68%. This invention leverages the microorganisms already present in the semi-coke phenol-ammonia wastewater. Without altering the original microbial community composition, it effectively improves the COD degradation rate through the synergistic effect of various strains, while also enhancing biological nitrogen and phosphorus removal. This invention has significant potential in reducing wastewater treatment costs and achieving wastewater purification and compliance with discharge standards.

[0075] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A complex microbial community, characterized in that, Including Acinetobacter sichuanensis, Pseudomonas songnenensis, and Diaphorobacter nitroreducens; The Acinetobacter sichuanensis mentioned is Acinetobacter sichuanensis LHH12, which was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232665. The *Pseudomonas songnenensis* strain mentioned is *Pseudomonas songnenensis* SJ2-76, which was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232667. The Diaphorobacter nitroreducens described is Diaphorobacter nitroreducens SYY4, which was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232669.

2. The composite microbial community according to claim 1, characterized in that, Also included is Thaurera aminoaromatica SJ2-12, which was deposited at the China Center for Type Culture Collection on December 25, 2023, with accession number CCTCC M 20232668.

3. A compound microbial agent, characterized in that, It contains the complex microbial community as described in claim 2.

4. The compound microbial agent as described in claim 3, characterized in that, The effective viable count ratio of Acinetobacter sichuanensis LHH12, Pseudomonas songnenensis SJ2-76, Thaurea aminoaromatica SJ2-12, and Diaphorobacter nitroreducens SYY4 was 1:1:(0.6-0.8):(0.6-0.8).

5. The application of the composite microbial flora as described in any one of claims 1-2 or the composite microbial agent as described in any one of claims 3-4 in the purification of semi-coke wastewater, wherein the phenol concentration in the semi-coke wastewater is ≥100 mg / L.

6. The application of the composite microbial community as described in any one of claims 1-2 or the composite microbial agent as described in any one of claims 3-4 in the degradation of chemical oxygen demand (COD) in semi-coke wastewater.

7. The application according to claim 5 or 6, characterized in that, The inoculation amount of the compound microbial flora or compound microbial agent is 0.8-1.2%.

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