A compound microbial agent, its preparation method and application

Abstract

The present invention discloses a composite microbial agent and its preparation method and application. The composite microbial agent includes bacterial strains, and the bacterial strains include Pseudomonas, Rhodococcus, and Bacillus. The Pseudomonas includes at least one of Pseudomonas sp. Z07 with a preservation number of CCTCC NO: M 20241294 and Pseudomonas sp. ZD1 with a preservation number of CCTCC NO: M 20241560. The Rhodococcus includes Rhodococcus sp. ZS1D-28 with a preservation number of CCTCC NO: M 2023986. The Bacillus includes Bacillus velezensis XN1 with a preservation number of CCTCC NO: M 2024857. Using the composite microbial agent of the present invention for the pollution remediation of slightly polluted water bodies in plain river network areas can grow and degrade nitrogen and phosphorus pollution in water bodies under a low-nutrient environment without adding an external carbon source or with less addition of a carbon source, and has the advantages of high efficiency, simplicity, and being conducive to popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms, and in particular to a composite bacterial agent capable of synergistically removing ammonia nitrogen, total nitrogen and total phosphorus, and a preparation method and application thereof. Background Art

[0002] In recent years, with the overload discharge of domestic, aquaculture and industrial wastewater, a large amount of nitrogen and phosphorus pollution has seriously threatened human health and the sustainable development of the ecosystem. Among the existing remediation technologies, microbial remediation has attracted widespread attention due to its economic applicability. Among them, directly adding functional bacteria to polluted water bodies is a recognized efficient and low-cost technical means.

[0003] In existing studies, most of the bacterial agents used for bioremediation are single strains. In actual applications, a single strain has limited effect on the removal of pollutants in water bodies. There are often problems such as unbalanced bacterial community structure, slow effect, and difficulty in reducing total nitrogen and total phosphorus content in water bodies. Composite microbial agents can overcome the shortcomings of physical and chemical methods and single microbial agent degradation methods due to their unique screening and domestication methods and appropriate compound ratios, and can achieve the best treatment effect, thereby controlling the cost of treatment and increasing the possibility of their application in the treatment of medium and small polluted water bodies. They have broad application prospects. In addition, denitrification in wastewater treatment is still at the level of ammonia nitrogen removal, and existing technologies and implementation standards rarely involve the removal of total nitrogen; in addition, total phosphorus removal is also mostly done by chemical methods, which increases costs and easily introduces external pollution, further deteriorating the environment.

[0004] Therefore, higher requirements are put forward for the degradation effect and effectiveness of existing water purification agents. In recent years, the discovery and system construction of aerobic denitrifying microorganisms and denitrifying phosphorus-accumulating microorganisms can improve the problems of nitrite and nitrate accumulation and low total phosphorus removal rate of traditional denitrifying agents. Providing a microbial agent with simple use method, significant water purification effect and synergistic pollution removal for the removal of ammonia nitrogen, total nitrogen and total phosphorus in natural water is an urgent problem to be solved by technicians in this field.

[0005] The patent specification with publication number CN115093986A discloses a composite bacterial agent for enhancing the denitrification and phosphorus removal performance of sewage, and its preparation method and application. The composite bacterial agent is a mixture of three strains of Pseudomonas stutzeri JUST-1, Paracoccuspantotrophus JUST-2, and Paracoccus versutus JUST-3, which can remove nitrogen and phosphorus at the same time.

[0006] The patent specification with the publication number CN110964664A discloses a composite bacterium agent for degrading N and P in aquaculture water bodies, its construction method and application. The composite bacterium agent is composed of Bacillus megaterium and Bacillus subtilis, and can be used for degrading ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and total phosphorus in the water body for crucian carp farming. Summary of the Invention

[0007] The present invention provides a composite bacterium agent, its preparation method and application. By utilizing the synergistic effect of different strains, while metabolizing the organic matter in the water body, it can remove ammonia nitrogen, total nitrogen and total phosphorus in the water body, playing a role in purifying the water quality.

[0008] In the first aspect, the present invention provides a composite bacterium agent, including bacterial strains, and the bacterial strains include Pseudomonas, Rhodococcus and Bacillus;

[0009] The Pseudomonas includes at least one of Pseudomonas sp. Z07 with the preservation number CCTCC NO: M 20241294 and Pseudomonas sp. ZD1 with the preservation number CCTCC NO: M 20241560;

[0010] The Rhodococcus includes Rhodococcus sp. ZS1D-28 with the preservation number CCTCC NO: M 2023986;

[0011] The Bacillus includes Bacillus velezensis XN1 with the preservation number CCTCC NO: M 2024857.

[0012] Pseudomonas sp. Z07 and Pseudomonas sp. ZD1 have the ability of simultaneous nitrification and denitrification and a complete denitrification metabolic pathway.

[0013] Rhodococcus sp. ZS1D-28 has significant polyphosphorus accumulation ability, but nitrite nitrogen accumulation will occur during the process of degrading nitrate nitrogen.

[0014] Bacillus velezensis XN1 has the function of degrading COD, and at the same time has functions such as rapid growth and resistance to environmental adversity, and can well adapt to the actual water body environment.

[0015] Pseudomonas sp. Z07, Pseudomonas sp. ZD1, Rhodococcus sp. ZS1D-28, and Bacillus velezensis XN1 can all be obtained from the China Center for Type Culture Collection (CCTCC). Among them, Rhodococcus sp. ZS1D-28 has been disclosed in the patent specification with the publication number CN117025476A in the applicant's previous application.

[0016] For the composite microbial agent described in the first aspect, the mass ratio of Pseudomonas sp. Z07 to Pseudomonas sp. ZD1 is preferably (5 - 10):(25 - 30).

[0017] For the composite microbial agent described in the first aspect, the mass ratio of the total mass of Pseudomonas sp. Z07 and Pseudomonas sp. ZD1 to Rhodococcus sp. ZS1D-28 and Bacillus velezensis XN1 is preferably (30 - 40):(40 - 50):(10 - 20).

[0018] In a preferred example, for the composite microbial agent described in the first aspect, the mass ratio of Pseudomonas sp. Z07, Pseudomonas sp. ZD1, Rhodococcus sp. ZS1D-28, and Bacillus velezensis XN1 is 5.9:29.1:46.6:18.4.

[0019] The composite microbial agent described in the first aspect may further include at least one of a carrier and a nutrient protectant.

[0020] The carrier may include at least one of wheat bran, montmorillonite powder, and soybean meal. Further, in the carrier, the mass ratio of the wheat bran, the montmorillonite powder, and the soybean meal may be (2 - 5):(1 - 4):(1 - 3), preferably 3:2:1, which is beneficial to improving the viable bacteria rate and the number of viable bacteria of the microbial agent.

[0021] The nutrient protectant may include at least one of skim milk powder, trehalose, glucose, and maltodextrin. Further, in the nutrient protectant, the mass ratio of the skim milk powder, the trehalose, the glucose, and the maltodextrin may be (1 - 4):(1 - 4):(1 - 4):(1 - 4), preferably 2:2:1:3, which is beneficial to improving the viable bacteria rate and the number of viable bacteria of the microbial agent.

[0022] In the compound bacterium agent described in the first aspect, the total viable count of Pseudomonas sp. Z07, Pseudomonas sp. ZD1, Rhodococcus sp. ZS1D-28, and Bacillus velezensis XN1 can reach not less than 1.0×10 11 CFU / g. This effect can be achieved by using the nutrient protectant described in the present invention. The nutrient protectant of the present invention can change the physical and chemical environment during the lyophilization of biological samples, reduce or prevent the damage to cells caused by lyophilization or rehydration, thereby reducing the frostbite mortality rate of cell proteins during the freezing process.

[0023] In the second aspect, the present invention provides a method for preparing the compound bacterium agent described in the first aspect, including: inoculating the bacterial strain seed liquid into a fermentation tank, adding a carrier, performing fermentation, stopping the fermentation during the logarithmic growth phase of the bacterial strain to obtain a fermentation broth, resuspending the wet bacterial cells obtained by centrifugal washing of the fermentation broth with an aqueous solution of the nutrient protectant, and then performing pre-freezing and lyophilization to obtain a high-density freeze-dried bacterial powder.

[0024] In the preparation method described in the second aspect, the bacterial strain seed liquid can be inoculated into the fermentation tank at 5% - 10% of the volume of the fermentation broth.

[0025] In the preparation method described in the second aspect, the carrier can be added at 0.1% - 0.2% g / mL of the volume of the fermentation broth.

[0026] In the preparation method described in the second aspect, the fermentation can be aerobic fermentation, the fermentation temperature can be 30 - 37°C, the fermentation pH can be 7.0 - 7.2, and the fermentation time can be 20 - 35 h.

[0027] In the preparation method described in the second aspect, the rotation speed of the centrifugation can be 5000 - 7000 rpm, preferably 6000 rpm.

[0028] In the preparation method described in the second aspect, the centrifugation time can be 5 - 20 min, preferably 15 min.

[0029] In the preparation method described in the second aspect, the mass ratio of the wet bacterial cells to the volume of the aqueous solution of the nutrient protectant can be 1 g:(1 - 8) mL, preferably 1 g:5 mL.

[0030] In the preparation method described in the second aspect, the mass fraction of the nutrient protectant in the aqueous solution of the nutrient protectant can be 5% - 10%.

[0031] In the preparation method described in the second aspect, the specific operation of the pre-freezing and lyophilization can include: pre-freezing at -18 - -22°C, preferably -20°C for 12 - 36 h, preferably 24 h, and then cooling to -55 - -60°C for vacuum freeze-drying.

[0032] The preparation method described in the second aspect can effectively reduce the death loss rate of bacterial cells during the freezing process. Compared with commercially available bacterial agents on the market, the viable count of the bacterial agent after freezing can be significantly increased (1 - 2 orders of magnitude), thereby greatly reducing the dosage of the bacterial agent in practical applications and reducing the cost of actual water body restoration projects.

[0033] In the third aspect, the present invention provides an application of the composite bacterial agent described in the first aspect in degrading nitrogen and phosphorus pollution in water bodies.

[0034] In the fourth aspect, the present invention provides a method for degrading nitrogen and phosphorus pollution in water bodies, including: adding the composite bacterial agent described in the first aspect to the water body to be treated for degrading nitrogen and phosphorus pollution.

[0035] The composite bacterial agent described in the present invention can be applied to river channels, aquaculture tail water, etc., for degrading ammonia nitrogen, total nitrogen, and total phosphorus pollution in water.

[0036] The composite bacterial agent can be first fully diluted with the actual water body and then added to the water body to be treated.

[0037] In some embodiments, the composite bacterial agent can be diluted with the water body to be treated such as river water, followed by aeration for 1 - 2 h and then evenly sprinkled into the water body to be treated.

[0038] The dosage of the composite bacterial agent described in the present invention can be 1 - 10 g / m 3 of the water body to be treated. For example, when the water body is a slightly polluted water body, the dosage of the composite bacterial agent can be 1 - 5 g / m 3 ; when the water quality is inferior to Class V, the dosage of the composite bacterial agent can be increased to 5 - 10 g / m 3 or even higher.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The present invention provides a microbial composite bacterial agent that can effectively remove total nitrogen and total phosphorus. It can be formed by mixed fermentation and freeze-drying of the screened Pseudomonas sp. Z07 and ZD1, Rhodococcus sp. ZS1D - 28, and Bacillus sp. XN1. Compared with commercially available river channel purification bacterial agents that can only remove ammonia nitrogen, the application effect of the composite bacterial agent described in the present invention is more extensive.

[0041] 2. The present invention screens out microorganisms that can adapt to the slightly polluted environment of natural water bodies through biological technology means such as natural domestication and directional breeding. Compared with commercially available river channel purification bacterial agents that require additional composite carbon sources or nutrient preparations, using the composite microbial bacterial agent of the present invention for the pollution repair of slightly polluted water bodies in plain river network areas can grow and degrade nitrogen and phosphorus pollution in water under a low-nutrient environment without adding carbon sources or adding less carbon sources, and has the advantages of high efficiency, simplicity, and being conducive to popularization and application.

[0042] 3. The present invention provides an application method of the composite microbial agent in the treatment of water bodies such as rivers. The microbial agent is diluted with on-site river water, which can fully mix the functional bacteria in the agent with the river water. Compared with the traditional method of directly spraying the microbial agent, while the purification efficiency of the agent is improved, the mixing of the agent can be made more thorough, avoiding the situation that the microbial strains concentrated hundreds or thousands of times during the preparation process cannot be evenly located throughout the water body. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Growth morphology of Pseudomonas sp. Z07 (a), Pseudomonas sp. ZD1 (b) and Bacillus velezensis XN1 (c) on the plate.

[0044] Figure 2 Degradation of single bacteria in a medium with a single nitrogen source (ammonia nitrogen, nitrate nitrogen or nitrite nitrogen). In the figure: a - c correspond to Rhodococcus sp. ZS1D - 28; d - f correspond to Pseudomonas sp. Z07; g - i correspond to Pseudomonas sp. ZD1.

[0045] Figure 3 3D response surface map of the nitrogen and phosphorus degradation rate of the interaction between bacteria. In the figure: a - d correspond to TN; e - h correspond to TP.

[0046] Figure 4 Physical photo of a composite microbial agent of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0048] Example 1 Isolation and Screening of Functional Bacteria

[0049] Pseudomonas sp. Z07, Pseudomonas sp. ZD1, and Bacillus velezensis XN1 were all screened from the river where the national control section of Zhu Feng Highway Bridge in Yaozhuang, Jiaxing is located. The culture and screening steps are as follows:

[0050] (1) Sample enrichment: Sampled from the river where the national control section of Zhu Feng Highway Bridge in Yaozhuang, Jiaxing is located, 100 mL of water sample was taken. After filtering the 100 mL water sample with a 0.22 μm filter membrane, the filter membrane was added to a 250 mL Erlenmeyer flask containing 100 mL of sterilized enrichment liquid medium, and cultured at 30 °C and 160 r / min for 48 h.

[0051] (2) Coating and screening: Dilute the enriched bacterial solution by gradient dilution method to a concentration of 10 -7 concentration, evenly coat it on the LB solid medium plate with a spiral inoculator and culture it. Incubate it in a biochemical incubator at 30°C for 48 h. The pH value of the LB solid medium plate is 7.0 - 7.2, the temperature is 28 - 37°C, and the components are: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract; 15 g / L agar powder. After the colonies form, select different colonies with different morphological colors from the LB solid medium for isolation.

[0052] (3) Sequencing and identification: Inoculate the single colony into the LB liquid medium, culture it at 30°C and 160 r / min for 24 h, and perform bacterial liquid PCR to amplify its 16S rDNA. Send the amplified product for sequencing analysis. Compare the sequencing results in the NCBI database and find that Z07 (SEQ ID NO: 1) and ZD1 (SEQ ID NO: 2) are Pseudomonas, and XN1 (SEQ ID NO: 3) is Bacillus. The isolated Pseudomonas Z07 and ZD1 and Bacillus XN1 are all deposited in the China Center for Type Culture Collection (CCTCC). The deposition times are June 19, 2024, July 15, 2024, and April 30, 2024 respectively. The deposition address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The strain deposit numbers are CCTCC NO: M20241294, CCTCC NO: M 20241560, and CCTCC NO: M 2024857 respectively.

[0053] (4) Effect verification: Select Z07 and XN1 from the LB solid medium and spot-inoculate them on the bromothymol blue (BTB) solid medium plate. Incubate them in a biochemical incubator at 30°C for 24 h, and observe the color change of the single colonies on the bromothymol blue plate. The results are as Figure 1 shown. After coating with Z07 and ZD1, the plate shows blue, indicating that denitrification occurs; after coating with XN1, the color change is not obvious, indicating that the denitrification ability of XN1 is weak.

[0054] The composition of the enrichment medium is: 3.44 g / L sodium citrate, 0.05 g / L KH 2 PO 4 , 1.00 g / L (NH 4 ) 2 SO 4 , 0.20 g / L MgSO 4 ·7H 2 O, 0.02 g / L K 2 SO 4 , 0.026 g / L CaCl 2 ·2H 2O, 1 mL / L trace element concentrate; the pH value of the trace element concentrate is 6.0 - 6.5, and the components are: 0.5 g / L FeCl 2 ·4H 2 O, 0.011 g / L MnCl 2 ·4H 2 O, 0.07 g / L ZnCl 2 , 0.006 g / L H 3 BO 3 , 0.036 g / L Na 2 MoO 4 ·2H 2 O, 0.014 g / L NiCl 2 ·6H 2 O, 0.002 g / L CuCl 2 ·2H 2 O。

[0055] The composition of the BTB solid screening medium is: 6.00 g / L sodium citrate, 0.05 g / L KH 2 PO 4 , 1.00 g / L (NH 4 ) 2 SO 4 , KNO 3 2.00 g, 0.20 g / L MgSO 4 ·7H 2 O, 0.02 g / L K 2 SO 4 , 0.026 g / L CaCl 2 ·2H 2 O, 1 mL 1% bromothymol blue solution, 15 g / L agar powder, 1 mL / L trace element concentrate; the pH value of the trace element concentrate is 6.0 - 6.5, and the components are: 0.5 g / L FeCl 2 ·4H 2 O, 0.011 g / L MnCl 2 ·4H 2 O, 0.07 g / L ZnCl 2 , 0.006 g / LH 3 BO 3 , 0.036 g / LNa 2 MoO 4 ·2H 2 O, 0.014 g / L NiCl 2 ·6H 2 O, 0.002 g / L CuCl 2 ·2H 2 O。

[0056] In the invention patent (application number 202311058533.0) previously applied for by the applicant, it has been confirmed that the strain ZS1D-28 has polyphosphate granules, and the average total phosphorus removal rate can reach 86.89% under ideal growth conditions.

[0057] Example 2 Verification of the Effects of Functional Bacteria

[0058] In this example, an artificial simulated wastewater containing a single nitrogen source was used as the experimental object to verify the nitrogen metabolic pathways of each strain, NH 4 + -N, NO 3 - -N, TP, and the initial concentrations of COD are shown in Table 1. Rhodococcus sp. ZS1D-28, Pseudomonas sp. Z07, and Pseudomonas sp. ZD1 were inoculated into a conical flask containing 1 L of simulated wastewater, and the concentration of the strain in the culture medium was maintained at OD600 = 0.01. The mixture was cultured at a constant temperature of 30 °C and 120 rpm for 2 d. The concentrations of nitrogen and phosphorus elements were detected every 12 h, and three parallel samples were set for each condition. The detection results of Z07, ZD1, and ZS1D-28 are as Figure 1 shown.

[0059] Table 1 Formulation of the Single Nitrogen Source Medium

[0060]

[0061] As shown in this example, ZS1D-28 has a good effect on degrading ammonia nitrogen and nitrate nitrogen. Within 48 h, the degradation rate is higher than 60% ( Figure 2 a, 2c), but it has no degradation effect on nitrite nitrogen ( Figure 2 b). In the invention patent (application number 202311058533.0) previously applied for by the applicant, it has been confirmed that this strain has polyphosphate granules, and the average total phosphorus removal rate can reach 86.89% under ideal growth conditions. As shown in this example, Z07 and ZD1 have excellent degradation effects. Through nitrogen balance analysis, it is verified that they can complete heterotrophic nitrification-aerobic denitrification. The ammonia nitrogen degradation rate is higher than 80% within 60 h ( Figure 2 d, 2f, 2g, 2i), and there is no accumulation of nitrite nitrogen during the denitrification process. The end product of metabolism is gaseous organic nitrogen ( Figure 2 e, 2h).

[0062] In this example, Bacillus velezensis XN1 has no degradation effect on nitrogen and phosphorus pollution, but it has the effects of rapid growth and resistance to environmental stress.

[0063] Example 3 Bacterial Agent Compound

[0064] The present invention provides a microbial complex bactericide that can be used for denitrification and phosphorus removal treatment of natural water bodies, aquaculture wastewater, etc., and the microbial complex bactericide can be applied in the field of water environment restoration.

[0065] Inoculate each strain into LB medium and culture it at 30 °C and 150 rpm for 20 - 30 h. Take an appropriate volume of the fermentation broth, centrifuge and resuspend it 3 times with 0.9% normal saline, and then add artificial simulated wastewater with a composite nitrogen source to verify the water purification effect of the bactericide for 24 h under different compounding ratio conditions. At this time, the initial OD of the artificial simulated wastewater 600 = 0.01, that is, OD 600-Z07 (A) + OD 600-ZS1D-28 (B) + OD 600-ZD1 (C) + OD 600-XN1 (D) = 0.01. The initial concentrations of NH 4 + -N, NO 3 - -N, TP, and COD in the simulated wastewater are shown in Table 2.

[0066] Table 2 Formulation of composite nitrogen source medium

[0067]

[0068] According to the D-optimal mixture design principle of Design-Expert 7.0 software, 20 simulated compounding combinations are formed. The compounding scheme and test results are shown in Table 3. Taking the total nitrogen (TN) degradation rate and total phosphorus (TP) degradation rate as the investigation indexes, the multiple regression equations between different compounding ratios of the strains and the degradation rates are simulated and fitted by the model recommended by Design-Expert 7.0 software. At the same time, the best compounding formula of the bactericide is obtained through calibration. The multiple regression equations of each factor with the TN degradation rate (Y1) and TP degradation rate (Y2) are as follows. The correlation coefficients R 2 values are 0.9922 and 0.9979 (> 0.90) respectively, and the adjusted determination coefficients R adj 2 values are 0.9753 and 0.9934 (> 0.90) respectively, indicating that the two equations have good fitting degrees, can reasonably analyze the change of response values, and have good repeatability.

[0069] Response value Y1 = 63.35A + 46.21B + 56.86C + 2.33D + 10.92AB + 20.88AC + 119.88AD + 7.56BC + 107.35BD + 79.23CD - 503.19ABC - 379.6ABD + 156.58ACD + 546.38BCD.

[0070] Response value Y2 = 17.41A + 86.88B + 10.51C + 14.12D + 118.03AB - 45.49AC - 1.81AD + 158.50BC + 53.54BD + 55.36CD + 1088.54ABC + 223.22ABD + 1628.49ACD - 50.05BCD.

[0071] Table 3 D - optimal mixture experiment design and results

[0072]

[0073] As can be seen from Table 4 and Table 5, for the fitting equation model, P < 0.001, indicating that the model difference is significant, and the lack - of - fit term is not significant (P > 0.05), indicating that the model equation fits well. The model can be used to predict the experimental results and optimize the ratio of the four strains. The interaction effects of AD, BD, CD, and ABC on the TN degradation rate are extremely significant (P < 0.001); the interaction effects of AB, AC, BD, CD, and ACD on the TP degradation are significant (P < 0.01), and the BC interaction effect is extremely significant (P < 0.001). The 3D response surface maps, i.e., contour lines, of the strain interaction effects on the TN and TP degradation rates are shown in Figure 3 . From Figure 3 it can be seen that the response surface formed by the interaction among factors ABCD on the degradation rate is a curved surface, indicating that there is a certain interaction among any three factors. For example, the response surface formed by the interaction among factors ACD on the TP degradation rate has the steepest slope, and the contour lines are elliptical, indicating that the interaction among ACD has a great influence on the TP degradation rate. This result is consistent with the result obtained from the variance analysis in Table 5. Combining the regression equation coefficients, the order of the influence of each strain on the TN degradation is A > C > B > D; the order of the influence of each strain on the TP degradation is B > A > D > C.

[0074] Table 4 Variance analysis of the TN degradation effect of the compound

[0075]

[0076] Note: * indicates a significant influence on the result (P < 0.05); ** indicates an extremely significant influence on the result (P < 0.01); *** indicates an extremely significant influence on the result (P < 0.001).

[0077] Table 5 Variance analysis of the TP degradation effect of the compound

[0078]

[0079] Note: * indicates a significant influence on the result (P < 0.05); ** indicates an extremely significant influence on the result (P < 0.01); *** indicates an extremely significant influence on the result (P < 0.001).

[0080] According to the D-optimal mixture model, the microbial composite bacterium agent described in this embodiment includes nitrifying bacteria, denitrifying bacteria and polyphosphate-accumulating bacteria such as Pseudomonas, Rhodococcus, and Bacillus. Among them, the specific Pseudomonas are Pseudomonas sp. Z07 and Pseudomonas sp. ZD1, and the preservation numbers are CCTCC NO: M 20241294 and CCTCC NO: M 20241560 respectively; the specific Rhodococcus is Rhodococcus sp. ZS1D-28, and the preservation number is CCTCC NO: M2023986; the specific Bacillus is Bacillus velezensis XN1, and the preservation number is CCTCC NO: M 2024857. The above strains were screened from polluted river water or purchased from the China Center for Type Culture Collection (CCTCC).

[0081] Among them, although Bacillus XN1 (D) does not have the characteristics of nitrogen and phosphorus degradation, the interaction between Pseudomonas Z07 (A), ZD1 (C) and Rhodococcus ZS1D-28 (B) is relatively significant. The reason is that Bacillus XN1 can decompose macromolecular organic matter in wastewater into small-molecular organic matter, thereby degrading COD in wastewater and providing a carbon source for the growth of other microorganisms; Pseudomonas Z07 (A) and ZD1 (C) can carry out nitrification and decompose polypeptide and ammonium salt substances in wastewater into nitrates; at the same time, Rhodococcus ZS1D-28 (B) and Pseudomonas have denitrifying phosphorus-accumulating and aerobic denitrification abilities respectively, so as to remove total phosphorus and total nitrogen in wastewater.

[0082] Preferably, the Bacillus includes Bacillus velezensis; the two Pseudomonas have the characteristics of heterotrophic nitrification-aerobic denitrification, and in particular, have a high-efficiency degradation effect on nitrite nitrogen; the denitrifying phosphorus-accumulating bacteria include Rhodococcus. It should be noted that Rhodococcus ZS1D-28 (B) can only carry out partial denitrification, and the reduction of nitrite is the rate-limiting step, which is prone to nitrite accumulation and is not conducive to the entire nitrogen and phosphorus degradation system. Therefore, after being compounded with Pseudomonas Z07 (A) and ZD1 (C), it can accelerate the reduction of nitrite nitrogen. Through the mutual cooperation of Bacillus, nitrifying bacteria, aerobic denitrifying bacteria and polyphosphate-accumulating bacteria, chemical oxygen demand, ammonia nitrogen, total nitrogen and total phosphorus in water can be effectively removed.

[0083] According to the analysis by response surface software, under the condition that the OD600 values of Z07:ZS1D-28:ZD1:XN1 are 0.0006:0.0047:0.0029:0.0018 (i.e., the mass percentages of the 4 kinds of bacteria in the compound bacterium agent are 5.9%:46.6%:29.1%:18.4%), the degradation rates of TN and TP are the highest, being 65.84% and 92.44% respectively. Under this condition, 3 groups of parallel verification tests were carried out. The actual TN degradation rate was 60.91±4.86%, and the actual TP degradation rate was 88.89±4.14%. The relative errors from the theoretical values were 4.93% and 3.55% respectively. Therefore, the D-optimal mixture response surface design is feasible.

[0084] Example 4

[0085] In this example, the slightly polluted river water body in a typical plain river network area was used as the experimental object. 10 L of water sample was taken, and the bacterium agent prepared according to the optimal formula of 4 kinds of bacteria determined in Example 3 was added. The compound bacterium agent includes a compound strain, a nutrient protectant, and a carrier. For the physical object of the bacterium agent, see Figure 4 . The dosage of the bacterium agent is 10 g / m 3 water body.

[0086] The preparation method of the compound bacterium agent is introduced as follows:

[0087] 1) Inoculate the bacterial strain seed liquid into the fermentation tank at 5% of the volume of the fermentation broth. Weigh a certain amount of carrier. The carrier is wheat bran, montmorillonite powder, and soybean meal with a mass ratio of 3:2:1. Add the carrier at 0.1% g / mL (1 g / L) of the volume of the fermentation broth. The fermentation temperature is 30 - 37 °C, the fermentation pH = 7.0, and aerobic fermentation is carried out for 35 h. Stop fermentation during the logarithmic growth phase of the bacterial strain to obtain the fermentation broth.

[0088] 2) Centrifuge the fermentation broth obtained in 1) at a medium and low speed. The centrifugation conditions are 6000 rpm for 15 min, and repeat centrifugation 3 times under the same conditions. Discard the supernatant, add an equal volume of 0.9% physiological saline to suspend and then centrifuge to obtain wet bacterial cells. The wet bacterial cells (bacterial sludge) are resuspended with an aqueous solution of a nutrient protectant in a certain ratio at a ratio of 1 g:5 mL to obtain a bacterial suspension. The total mass fraction of the compound nutrient protectant in the aqueous solution of the nutrient protectant is 10%.

[0089] 3) Turn on the freeze dryer and pre-freeze for about 5 minutes until the temperature drops to -55 °C. Put the pre-frozen sample pre-frozen at -20 °C for 24 h into the freeze dryer at -55 °C for vacuum freeze-drying, and obtain the compound bacterial powder after drying.

[0090] The measurement results show that within 48 hours after adding the bacterial powder, the ammonia nitrogen content of the self-made composite bactericide decreased from 2.03 mg / L before dosing to 0.56 mg / L after dosing, the total nitrogen decreased from 5.54 mg / L before dosing to 2.76 mg / L after dosing, and the total phosphorus decreased from 0.40 mg / L before dosing to 0.18 mg / L after dosing. The water quality after restoration reached the Class III water quality standard of the Surface Water Environment Quality Standard. Table 6 shows the purification effect (removal rate) of the composite bactericide on river sewage within 48 hours.

[0091] Table 6

[0092]

[0093] As can be seen from the above-mentioned implementation cases, the strains in the bactericide of the present invention are more suitable for slightly polluted environments, and in the river, Bacillus, nitrifying bacteria, and denitrifying polyphosphate-accumulating bacteria each play their roles, and can synergistically promote the degradation of water pollutants and ultimately achieve the purpose of water purification.

[0094] In summary, the present invention has compounded 4 strains with different nitrogen and phosphorus metabolic pathways through the D-optimal mixture model. Compared with single strains, the water purification effect is significantly improved, and it can simultaneously and synergistically remove substances such as nitrogen, phosphorus, and COD in the water body. In addition, through the synergistic protection of skim milk powder, trehalose, glucose, and maltodextrin, the viable bacteria count and viable bacteria rate of the bactericide can be greatly increased at a relatively low concentration of the protective agent, and various original physiological and biochemical characteristics and biological activities can be maintained as much as possible during the freeze-drying process, making it not only convenient for storage and transportation during actual application, but also reducing the engineering application cost by increasing the number of viable bacteria.

[0095] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A composite bacterial agent, comprising bacterial species, characterized in that: The bacterial species are composed of Pseudomonas sp., Rhodococcus sp. and Bacillus sp.; The Pseudomonas is at least one of Pseudomonas sp.Z07 with a deposit number of CCTCC NO: M 20241294 and Pseudomonas sp.ZD1 with a deposit number of CCTCC NO: M 20241560; The rhodococcus is Rhodococcus sp. ZS1D-28 with a deposit number of CCTCC NO: M 2023986; The bacillus is Bacillus velezensis XN1 with a deposit number of CCTCC NO: M 2024857.

2. The composite bacterial agent according to claim 1, characterized in that The mass ratio of Pseudomonas sp. Z07 and Pseudomonas sp. ZD1 is 5-10:25-30; The mass ratio of the total mass of Pseudomonas sp.Z07 and Pseudomonas sp.ZD1 to that of Rhodococcus sp.ZS1D-28 and Bacillus velezensis XN1 is 30-40:40-50:10-20.

3. The composite bacterial agent according to claim 2, characterized in that: The mass ratio of Pseudomonas sp.Z07, Pseudomonas sp.ZD1, Rhodococcus sp.ZS1D-28 and Bacillus velezensis XN1 is 5.9:29.1:46.6:18.

4.

4. The composite bacterial agent according to any one of claims 1 to 3, characterized in that: The composite bacterial agent also includes at least one of a carrier and a nutrient protective agent; The carrier comprises at least one of wheat bran, montmorillonite powder and soybean meal; The nutritional protective agent includes at least one of skimmed milk powder, trehalose, glucose and maltodextrin.

5. The composite bacterial agent according to claim 4, characterized in that: In the carrier, the mass ratio of the wheat bran, the montmorillonite powder and the soybean meal is 2-5:1-4:1-3.

6. The composite bacterial agent according to claim 5, characterized in that: In the carrier, the mass ratio of the wheat bran, the montmorillonite powder and the soybean meal is 3:2:

1.

7. The composite bacterial agent according to claim 4, characterized in that: In the nutritional protective agent, the mass ratio of the skimmed milk powder, the trehalose, the glucose and the maltodextrin is 1-4:1-4:1-4:1-4.

8. The composite bacterial agent according to claim 7, characterized in that: In the nutritional protective agent, the mass ratio of the skimmed milk powder, the trehalose, the glucose and the maltodextrin is 2:2:1:

3.

9. The composite bacterial agent according to claim 1, characterized in that: In the composite bacterial agent, the total number of live bacteria of Pseudomonas sp. Z07, Pseudomonas sp. ZD1, Rhodococcus sp. ZS1D-28 and Bacillus velezensis XN1 is not less than 1.0×10 11 CFU / g.

10. The method for preparing the composite bacterial agent according to any one of claims 1 to 9, characterized in that: include: The bacterial seed liquid is inoculated into a fermentation tank, a carrier is added, and fermentation is carried out. The fermentation is stopped during the logarithmic growth phase of the bacterial strain to obtain a fermentation liquid. The wet bacteria obtained by centrifuging and washing the fermentation liquid are resuspended in an aqueous solution of a nutrient protective agent, pre-frozen, and freeze-dried to obtain a high-density freeze-dried bacterial powder.

11. The preparation method according to claim 10, characterized in that: The bacterial seed liquid is inoculated into the fermentation tank at 5% to 10% of the volume of the fermentation liquid; The carrier is added at 0.1% to 0.2% g / mL of the fermentation liquid volume; The fermentation is aerobic fermentation, the fermentation temperature is 30-37°C, the fermentation pH is 7.0-7.2, and the fermentation time is 20-35h; The centrifugal speed is 5000-7000 rpm; The centrifugation time is 5 to 20 minutes; The ratio of the mass of the wet bacteria to the volume of the aqueous solution of the nutrient protective agent is 1 g: 1 to 8 mL; The mass fraction of the nutrient protective agent in the aqueous solution of the nutrient protective agent is 5% to 10%; The specific operation of pre-freezing and freeze-drying includes: pre-freezing at -18 to -22°C for 12 to 36 hours, then cooling to -55 to -60°C, and performing vacuum freeze-drying.

12. The preparation method according to claim 11, characterized in that: The centrifugal rotation speed is 6000 rpm.

13. The preparation method according to claim 11, characterized in that: The centrifugation time is 15 min.

14. The preparation method according to claim 11, characterized in that: The ratio of the mass of the wet bacteria to the volume of the aqueous solution of the nutrient protective agent is 1 g:5 mL.

15. The preparation method according to claim 11, characterized in that: The specific operation of pre-freezing and freeze-drying includes: pre-freezing at -20°C for 24 hours, then cooling to -55 to -60°C, and performing vacuum freeze-drying.

16. Use of the composite bacterial agent according to any one of claims 1 to 9 in degrading nitrogen and phosphorus pollution in water bodies.

17. A method for degrading nitrogen and phosphorus pollution in water, characterized in that: include: The composite bacterial agent according to any one of claims 1 to 9 is added to the water body to be treated to degrade nitrogen and phosphorus pollution.

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