Liquid live bacteria preservation method of thiobacillus denitrificans

Abstract

This invention relates to the field of applied microbiology technology and discloses a method for liquid live preservation of *Thiobacillus denitrificationus*, comprising the following steps: inoculating *Thiobacillus denitrificationus* into a fermentation medium and culturing it, followed by a displacement treatment to obtain a sulfur autotrophic denitrification rate of not less than 500 mg N·L. ‑1 ·h ‑1 The original bacterial culture is prepared by centrifugation or membrane concentration, followed by replacement with a replacement medium containing a compound sulfur source and potassium nitrate, wherein the compound sulfur source is a combination of a soluble sulfur source and a slow-release sulfur source. The treated bacterial culture is then stored at a specific temperature, maintaining the system in an oxidized state during storage. This method for preserving live liquid *Thiobacillus denitrifyingus* aims to address the problem that *Thiobacillus denitrifyingus* cannot be preserved long-term as a liquid bacterial agent at room temperature.

Description

Technical Field

[0001] This invention relates to the field of applied microbiology technology, specifically to a method for preserving live denitrifying bacteria (Thiobacillus denitrification) in liquid form. Background Technology

[0002] Thiobacillus denitrifyingus is an important microbial species that plays a significant role in protecting the ecological environment and treating nitrate wastewater. Nitrate wastewater treatment is a crucial aspect of environmental protection, and Thiobacillus denitrifyingus is the main microorganism used to treat nitrate wastewater, playing a positive role in desulfurization and denitrification projects. Under anaerobic conditions, Thiobacillus denitrifyingus can utilize sodium thiosulfate, elemental sulfur, and sulfides as electron donors to reduce nitrates to nitrogen gas, thus achieving the purpose of nitrate removal.

[0003] In the field of wastewater treatment, traditional biological denitrification technology mainly relies on heterotrophic denitrifying microorganisms. These microorganisms consume a large amount of organic carbon sources during the denitrification process. Thiobacillus denitrification, as an autotrophic microorganism, can remove nitrates through sulfur autotrophic denitrification, achieving deep denitrification. However, because Thiobacillus denitrification mainly uses sulfur as an electron donor for denitrification, in an anaerobic environment, its preservation in liquid form easily generates insoluble sulfides and hydrogen sulfide, making the entire liquid system black and smelly. Currently, there is relatively little research on the preservation of live autotrophic microorganisms both domestically and internationally; most studies focus on immobilization and freeze-drying. For example, patent (publication number CN111944799B) discloses a method for preparing and applying immobilized particles containing Thiobacillus denitrification. This patent obtains an embedding body by mixing Thiobacillus denitrification with oyster shell powder, obtains an embedding agent by heating polyvinyl alcohol solution, sulfur, and water, and adds a crosslinking agent to obtain immobilized particles of Thiobacillus denitrification. These particles can effectively remove NO3 from wastewater with a low C / N ratio. - -N. For example, patent (publication number CN109266590A) discloses a denitrifying compound bacterial agent, its preparation method, and its application. This patent mixes multiple bacterial solutions, one of which is *Thiobacillus denitrifyingus* (18-23%), then mixes it with an adsorbent and freeze-dries it, effectively removing nitrogenous compounds from wastewater. Furthermore, patent (publication number CN113122465A) discloses a method for preparing and preserving nitrifying bacteria dry powder. This patent concentrates nitrifying bacteria solution, adds a protective agent, and vacuum-dries it to produce nitrifying bacteria dry powder. After long-term preservation, the activity recovers quickly and with a high activity recovery rate. Summary of the Invention

[0004] The purpose of this invention is to solve the problem that *Thiobacillus denitrification* as a liquid inoculant cannot be preserved for a long time at room temperature, and to propose a method for preserving live liquid *Thiobacillus denitrification*.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] A method for preserving live denitrifying thiobacillus in liquid comprises the following steps:

[0007] S10. Inoculate *Thiobacillus denitrificationus* into fermentation medium and culture. After replacement treatment, obtain a sulfur autotrophic denitrification rate of not less than 500 mg N·L. -1 ·h -1 The original bacterial culture;

[0008] S20. After centrifuging or membrane concentrating the original bacterial solution, it is replaced with a replacement medium containing a compound sulfur source and potassium nitrate. The compound sulfur source is a combination of a soluble sulfur source and a slow-release sulfur source.

[0009] S30. The bacterial culture treated with S20 is stored at a specific temperature, and the system is kept in an oxidized state during the storage process.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, in S10, the fermentation medium comprises: 0.5-1 g / L NH4Cl, 0.5-1.5 g / L MgSO4·7H2O, 3-5 g / L KH2PO4, 8-12 g / L Na2S2O3·5H2O, 3-6 g / L KNO3, 1-3 g / L NaHCO3, 0.005-0.015 g / L FeSO4·7H2O, and 0.5-1.5 mL / L trace elements. The pH is 6.5-8.0; the trace elements include EDTA-2Na at 50 g / L, CaCl2·2H2O at 7.34 g / L, FeSO4·7H2O at 5.0 g / L, MnCl2·4H2O at 2.5 g / L, ZnSO4·7H2O at 2.2 g / L, (NH4)6Mo7O2·4H2O at 0.5 g / L, CaSO4·5H2O at 0.2 g / L and NaOH at 11.0 g / L.

[0012] Furthermore, in S10, the denitrifying thiobacillus used is a sulfur-autotrophic denitrifying bacterium T. h i ob aci ll usdenitrificans SZG-SAD-004, preservation number CCTCCNO: M2023992; the inoculum of this strain was 10%, and the original bacterial solution was obtained after two replacement treatments. Both replacement treatments were achieved by removing the metabolites produced during fermentation to avoid the inhibition of the activity of denitrifying thiobacilli by the metabolites, and to ensure that the sulfur autotrophic denitrification rate of the original bacterial solution was stably maintained above 500 mg N·L-1·h-1.

[0013] Furthermore, in step S20, the replacement ratio is 40%-100%, where the replacement ratio is the volume ratio of the replacement culture medium to the concentrated bacterial solution; the centrifugation speed is 3000-5000 r / min, and the centrifugation time is 10-20 min; the membrane concentration uses an ultrafiltration membrane with a molecular weight cutoff of 10000-30000 Da, and the operating pressure is 0.1-0.3 MPa.

[0014] Furthermore, in S20, the soluble sulfur source in the compound sulfur source is Na2S2O3·5H2O, with a concentration of 10-15 g / L; the slow-release sulfur source is elemental sulfur or FeS mineral powder, with a concentration of 10-20 g / L.

[0015] Furthermore, in S20, the components of the replacement medium are: MgCl·6H2O 0.5-1.5 g / L, KH2PO4 1-3 g / L, KNO3 10-15 g / L, NaHCO3 3-6 g / L, FeSO4·7H2O 0.005-0.015 g / L, and a compound sulfur source 20-35 g / L, and the pH of the replacement medium is adjusted to 7.0-7.5.

[0016] Furthermore, in step S30, the system is maintained in an oxidized state by introducing oxygen into the bacterial solution, the amount of oxygen depending on the preservation system, requiring 2-3 L of pure oxygen per liter of bacterial solution; or by adding an oxidizing agent, namely H2O2, at an amount of 0.01%-0.05% of the bacterial solution volume.

[0017] Furthermore, in S30, when the specific temperature is 4°C, the storage time can reach more than 90 days, and the loss rate of sulfur autotrophic denitrification rate of the bacterial solution after 90 days of storage does not exceed 30%; when the specific temperature is 28°C, the storage time is about 30 days, and the loss rate of sulfur autotrophic denitrification rate of the bacterial solution after 30 days of storage does not exceed 50%.

[0018] Furthermore, in S30, the storage container is a sealed glass or plastic container, with 10%-20% of the headspace volume reserved inside the container to balance the pressure changes of the system during storage.

[0019] Compared with the prior art, the technical solution of this application has the following beneficial technical effects:

[0020] This invention employs a replacement culture medium containing a compound sulfur source and potassium nitrate. The soluble sulfur source in the compound sulfur source meets the sulfur requirements of microorganisms in the early stages of preservation, while the slow-release sulfur source gradually releases sulfur to maintain the electron donor requirements of denitrifying thiobacilli. Simultaneously, the presence of potassium nitrate provides electron acceptors for the microorganisms, maintaining the system in an oxidized state during preservation, preventing sulfate reduction to sulfides, and further inhibiting hydrogen sulfide production. This fundamentally avoids the occurrence of black and foul-smelling liquid systems. Furthermore, it yields original bacteria with a sulfur autotrophic denitrification rate of not less than 500 mg N·L⁻¹·h⁻¹. This liquid preservation method, combined with replacement treatment, allows the bacterial culture to maintain high activity without the need for activation as in methods like freeze-drying. It can directly act on the device to remove total nitrogen. Furthermore, the process is simple, requiring only the replacement of the original bacterial culture medium. Compared to complex processes such as embedding and immobilization, it is much easier to operate. In addition to the culture medium, only a certain amount of oxidizing substances are needed to maintain the preservation conditions, significantly reducing costs. Moreover, this preservation method can be directly used for the liquid preservation of Thiobacillus denitrification agent products, expanding its application scenarios and meeting the needs of actual production for agent preservation. Attached Figure Description

[0021] Figure 1 This invention relates to the rate change of Td and the loss rate at each stage within 90 days at 4℃.

[0022] Figure 2 This invention relates to the rate change of Td and the loss rate at each stage within 30 days at 28℃. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention aims to achieve long-term preservation of liquid live bacteria Thiobacillus denitrificans SZG-SAD-004, with accession number CCTCCNO: M2023992, on June 12, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The preservation method aims to maintain high bacterial activity while avoiding a black and foul-smelling system. Furthermore, the preserved bacterial solution can be directly applied to nitrate wastewater treatment without activation.

[0025] Strains preparation and fermentation culture

[0026] strain selection

[0027] The denitrifying thiobacillus used in this preservation method is a sulfur-autotrophic denitrifying bacterium. o The strain *Bacillus denitrificans* SZG-SAD-004, with accession number CCTCCNO: M2023992, possesses highly efficient sulfur autotrophic denitrification capabilities. Under suitable conditions, it can rapidly convert nitrates into nitrogen gas, making it an excellent strain for treating nitrate wastewater.

[0028] Fermentation culture medium preparation

[0029] The fermentation medium consists of the following components: NH4Cl 0.5-1 g / L, MgSO4·7H2O 0.5-1.5 g / L, KH2PO4 3-5 g / L, Na2S2O3·5H2O 8-12 g / L, KNO3 3-6 g / L, NaHCO3 1-3 g / L, FeSO4·7H2O 0.005-0.015 g / L, and trace elements 0.5-1.5 mL / L, with a pH of 6.5-8.0.

[0030] The composition of trace elements strictly adheres to the following specifications: EDTA-2Na 50 g / L, CaCl2·2H2O 7.34 g / L, FeSO4·7H2O 5.0 g / L, MnCl2·4H2O 2.5 g / L, ZnSO4·7H2O 2.2 g / L, (NH4)6Mo7O2·4H2O 0.5 g / L, CaSO4·5H2O 0.2 g / L, and NaOH 11.0 g / L. During preparation, each component is dissolved separately first, then mixed according to the specified ratio. Finally, the pH is adjusted to the designated range with hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized at high temperature (121℃, 20 min) before use.

[0031] Inoculation and Culture

[0032] The above-mentioned *Thiobacillus denitrificationus* strain was inoculated into the sterilized fermentation medium at a 10% inoculum. Inoculation must be performed under aseptic conditions, preferably using a flame ring inoculation method to avoid contamination. After inoculation, the culture was carried out in anoxic conditions in a fermenter, with the sulfur autotrophic denitrification rate of the bacterial solution monitored in real time during the culture.

[0033] Replacement treatment and acquisition of original bacterial culture

[0034] Two replacement treatments are required during cultivation to remove metabolic products generated during fermentation and prevent these products from inhibiting the activity of *Thiobacillus denitrifyingus*. The first replacement is performed at the early stage of the logarithmic growth phase, using fresh culture medium with the same composition as the fermentation medium, with a replacement volume of 50% of the original bacterial volume. The second replacement is performed in the middle of the logarithmic growth phase, with a replacement volume of 60%-70% of the original bacterial volume.

[0035] After the replacement operation is completed, the sulfur autotrophic denitrification rate of the bacterial solution needs to be tested to ensure that it is not less than 500 mg N·L. -1 ·h -1 The detection method uses the nitrate ion electrode method. 100 mL of bacterial culture is taken, and a certain amount of KNO3 solution (final concentration 50 mg / L) is added. The mixture is reacted at 28℃ for 1 hour, and the amount of nitrate removed is calculated to obtain the sulfur autotrophic denitrification rate. If the rate does not meet the target, cultivation continues and a third replacement (50% replacement volume) is performed until the rate meets the requirements. The bacterial culture obtained at this point is the original bacterial culture.

[0036] Bacterial culture concentration and replacement of culture medium

[0037] Concentration of original bacterial culture

[0038] The obtained original bacterial culture is then centrifuged or concentrated using a membrane. If centrifugation is used, the centrifugation parameters are set to 3000-5000 rpm for 10-20 minutes. Specifically, a suitable centrifuge can be selected based on the volume of the bacterial culture. For cultures under 1L, a benchtop centrifuge is used at 4000 rpm for 15 minutes; for cultures over 1L, a large-capacity centrifuge is used at 3500 rpm for 20 minutes. After centrifugation, the supernatant is discarded, and the bottom bacterial sludge is retained. The volume of the sludge should be approximately 10%-20% of the original bacterial culture volume.

[0039] If membrane concentration is used, select an ultrafiltration membrane with a molecular weight cutoff of 10,000-30,000 Da, and control the operating pressure at 0.1-0.3 MPa. During concentration, maintain a membrane flow rate of 1-2 m / s to reduce membrane fouling. Check the volume of the concentrate every 30 minutes. Stop operation when the concentrate reaches 10%-20% of its original volume and collect the concentrated bacterial solution.

[0040] Preparation of replacement culture medium

[0041] The replacement culture medium consists of 0.5-1.5 g / L MgCl·6H2O, 1-3 g / L KH2PO4, 10-15 g / L KNO3, 3-6 g / L NaHCO3, 0.005-0.015 g / L FeSO4·7H2O, and 20-35 g / L of a compound sulfur source. The compound sulfur source is a combination of a soluble sulfur source and a slow-release sulfur source, specifically 10-15 g / L Na2S2O3·5H2O (soluble sulfur source) and 10-20 g / L elemental sulfur or FeS mineral powder (slow-release sulfur source). Elemental sulfur is preferred as a slow-release sulfur source because when the combined sulfur source is a combination of 10-15 g / L Na₂S₂O₃·5H₂O and 15-20 g / L elemental sulfur, the sulfur autotrophic denitrification rate of the bacterial culture can still be maintained at 260 mg N·L after storage at 28℃ for 30 days. -1 ·h -1 The above conditions were met, and no black or foul-smelling substances were observed in the system.

[0042] During preparation, first dissolve all components except the compound sulfur source in deionized water, adjust the pH to 7.0-7.5 with hydrochloric acid or sodium hydroxide solution, then add the weighed compound sulfur source, stir until Na2S2O3·5H2O is completely dissolved (elemental sulfur or FeS mineral powder can be in suspension), and finally sterilize at high temperature (121℃, 20min), and cool to room temperature for later use.

[0043] Displacement operation

[0044] According to the preset replacement ratio (volume ratio of replacement medium to concentrated bacterial solution), mix the concentrated bacterial solution with the replacement medium. The replacement ratio can be selected from 40% to 100%, depending on the storage time and temperature: if the plan is to store at 4℃ for more than 90 days, choose a replacement ratio of 80%-100%; if the plan is to store at 28℃ for about 30 days, choose a replacement ratio of 40%-60%.

[0045] The operating steps are as follows: ① Determine the volume of concentrated bacterial solution (V1) and the replacement ratio (R), and calculate the required replacement medium volume (V2 = V1 × R); ② Add the concentrated bacterial solution to a sterile conical flask or storage tank; ③ Add the replacement medium according to the calculated volume, and stir evenly with a sterile glass rod (for storage tanks, the stirring device can be turned on at a speed of 50-100 r / min for 5-10 min); ④ Take a sample to test the bacterial solution concentration, ensuring it is within the range of 0.8-1.2. If the concentration is too high, the replacement medium volume can be increased appropriately; if the concentration is too low, it can be adjusted by centrifugation (3000 r / min, 10 min) to concentrate.

[0046] Preservation and maintenance of bacterial culture

[0047] Preparation of storage containers

[0048] Choose a sealed glass or plastic container as the storage container. The container capacity is determined according to the volume of the bacterial solution, typically a 500mL, 1L, or 5L wide-mouth bottle. Ensure the container is clean and sterile (this can be achieved through autoclaving at 121°C for 20 minutes). Before filling with the bacterial solution, check the container's seal to ensure there are no leaks. When filling with the bacterial solution, leave 10%-20% of the headspace volume to balance pressure changes in the system during storage. For example, a 500mL container should hold a maximum of 400-450mL of bacterial solution.

[0049] Storage temperature control

[0050] The replaced bacterial culture was stored at a specific temperature. If stored at 4℃, the container could be placed in the refrigerator's crisper drawer, with temperature fluctuations controlled within ±1℃. Under these conditions, the storage time could reach over 90 days, and the sulfur autotrophic denitrification rate loss of the bacterial culture after 90 days of storage would not exceed 30%. If stored at 28℃, the container could be placed in a constant temperature incubator, with temperature fluctuations controlled within ±2℃. Under these conditions, the storage time would be approximately 30 days, and the sulfur autotrophic denitrification rate loss of the bacterial culture after 30 days of storage would not exceed 50%.

[0051] During storage, the temperature needs to be recorded daily. If the temperature exceeds the set range, the equipment parameters should be adjusted promptly.

[0052] Maintenance of the oxidation state of the system

[0053] During preservation, the system must be kept in an oxidized state. This can be achieved using the following two methods:

[0054] Method 1: Introduce oxygen into the bacterial culture. Use a small air pump connected to the bottom of the preservation container (with an aeration stone installed at the inlet) via sterile silicone tubing. Set the oxygenation rate to 0.1-0.5 L / (L·h). Depending on the preservation system, 2-3 L of oxygen should be introduced per liter of bacterial culture. For example, for 500 mL of bacterial culture, set the oxygenation rate to 0.3 L / (L·h) and oxygenate for 2 hours. During the oxygenation process, the container lid can be slightly opened (leaving a gap) to release gas from the system. After opening the lid, oxygen must be introduced again.

[0055] Method 2: Add an oxidizing agent. Choose H2O2 as the oxidizing agent, adding it at 0.01%-0.05% of the bacterial culture volume. To do this, dilute a 30% H2O2 solution 100 times with sterile water, then add the calculated amount to the bacterial culture, gently shaking the container to mix thoroughly. For example, add 0.1-0.25 mL of diluted H2O2 solution to 500 mL of bacterial culture, repeating this process every 3 days.

[0056] The choice between the two methods is based on the following criteria: For storage at 4℃, oxygenation is preferred to avoid the potential impact of slow H2O2 decomposition on the bacterial cells at low temperatures; for storage at 28℃, adding H2O2 is preferred due to its simpler operation. Regardless of the method used, the redox potential (ORP) of the system must be monitored after opening the container to ensure it remains between 0-100mV. If the ORP is below 0mV, the oxygenation time or the amount of H2O2 added needs to be increased.

[0057] Regularly monitor the state and activity of the bacterial solution: ① Visual inspection: Observe the bacterial solution daily for any black or foul odor. If the system turns black or has a hydrogen sulfide odor, immediately increase measures to maintain the oxidation state (e.g., extend the aeration time to 5-6 hours); ② Activity testing: Detect the sulfur autotrophic denitrification rate every 15 days (stored at 4℃) or every 10 days (stored at 28℃). If the rate loss exceeds the preset value (not exceeding 30% within 90 days of storage at 4℃), such as... Figure 1 As shown in Table 1 below, no more than 50% of the samples stored at 28℃ for 30 days should be affected. Figure 2 (As shown in Table 2 below), it needs to be treated promptly (e.g., for wastewater treatment or for replacement treatment).

[0058]

[0059]

[0060] Table 1 shows the rate change of Td and the loss rate values ​​at each stage within 90 days at 4℃.

[0061]

[0062] Table 2 shows the rate change of Td and the loss rate values ​​at each stage within 30 days at 28℃.

[0063] In addition, the start time of preservation should be recorded, and the expiration time should be estimated based on the preservation temperature, so as to prepare in advance for the use of the bacterial solution or for renewal of preservation.

[0064] Advantages of preservation methods

[0065] Through the synergistic effect of each step, the problem of liquid preservation of *Thiobacillus denitrifyingus* in existing technologies is effectively solved. The entire process can be completed simply by replacing the original bacterial solution with a replacement culture medium, making the process simple. The preserved bacterial solution maintains high activity throughout and can be directly used in wastewater treatment devices to quickly remove total nitrogen without activation. The cost is low, requiring only an air pump or a small amount of H2O2 to maintain the oxidation state of the system, in addition to the culture medium. Furthermore, it can be directly applied to the liquid preservation of *Thiobacillus denitrifyingus* inoculant products, providing a feasible solution for their industrial production and application.

[0066] By following the detailed steps outlined above, it is possible to effectively preserve *Thiobacillus denitrificationus* for more than 90 days at 4℃ and for about 30 days at 28℃. During the preservation period, the bacterial solution does not exhibit blackening or foul odor, and the activity loss rate is controlled within a reasonable range, fully meeting the preservation requirements of liquid *Thiobacillus denitrificationus* inoculants.

[0067] Example

[0068] The following are the actual applications of the preserved bacterial solution in nitrate-containing wastewater:

[0069] Step 1: Replace 80% of the bacterial culture with a sulfur autotrophic denitrification rate of 500 or higher using a replacement medium. The replacement medium consists of the following components: MgCl·6H2O 0.5-1.5 g / L, KH2PO4 1-3 g / L, KNO3 10-15 g / L, NaHCO3 3-6 g / L, FeSO4·7H2O 0.005-0.015 g / L, and a compound sulfur source of 20-35 g / L. The compound sulfur source is: 10-15 g / L Na2S2O3·5H2O + 10-20 g / L elemental sulfur / FeS mineral powder, with elemental sulfur being preferred. After replacement, store at 28℃ for 30 days before removing.

[0070] Step 2: Inoculate the bacterial culture stored for 30 days into a sulfur autotrophic pilot-scale device to verify its practical application effect. No activation is required; inoculation is performed directly. Inoculate 1% of the culture into the pilot-scale device containing the sulfur autotrophic carrier. After introducing simulated wastewater, allow the culture to stand for 24 hours. The total nitrogen level drops to 20 mg / L, indicating the culture is complete. First stage: Introduce simulated wastewater with a retention time of 2 hours; the effluent total nitrogen level is less than 15 mg / L. Second stage: After 12 hours, increase the nitrate concentration in the simulated wastewater and decrease the retention time to 1 hour; the effluent total nitrogen level is less than 15 mg / L. Third stage: After 24 hours, increase the nitrate concentration in the simulated wastewater and decrease the retention time to 0.5 hours; the effluent total nitrogen level is less than 15 mg / L.

[0071] It will then be able to run stably, as shown in Table 3.

[0072]

[0073]

[0074] Table 3 shows the actual application effects of Td after room temperature storage.

[0075] The following are the compositions of the simulated wastewater at each stage: ① Cultivation stage: 100 mg / L nitrate, 2 mg / L total phosphorus; ② First stage: 100 mg / L nitrate, 2 mg / L total phosphorus; ③ Second stage: 300 mg / L nitrate, 2 mg / L total phosphorus; ④ Third stage: 500 mg / L nitrate, 2 mg / L total phosphorus.

[0076] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0077] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preserving live denitrifying bacteria in liquid, characterized in that, Includes the following steps: S10. Inoculate *Thiobacillus denitrificationus* into fermentation medium and culture. After replacement treatment, obtain a sulfur autotrophic denitrification rate of not less than 500 mg N·L⁻¹. -1 ・h -1 The original bacterial culture; S20. After centrifuging or membrane concentrating the original bacterial culture, a replacement medium is used for replacement. The replacement medium contains a compound sulfur source and potassium nitrate. The compound sulfur source is a combination of a soluble sulfur source and a slow-release sulfur source. In the compound sulfur source, the soluble sulfur source is Na2S2O3・5H2O, with a concentration of 10-15 g / L; the slow-release sulfur source is elemental sulfur or FeS mineral powder, with a concentration of 10-20 g / L. The components of the replacement medium are: MgCl2・6H2O 0.5-1.5 g / L, KH2PO4 1-3 g / L, KNO3 10-15 g / L, NaHCO3 3-6 g / L, FeSO4・7H2O 0.005-0.015 g / L, and the compound sulfur source 20-35 g / L. The pH of the replacement medium is adjusted to 7.0-7.

5. S30. The bacterial solution treated in S20 is stored at a specific temperature. During the storage process, the system is kept in an oxidized state. In S30, the system is kept in an oxidized state by introducing oxygen into the bacterial solution. The amount of oxygen introduced depends on the storage system. 2-3L of pure oxygen is required per liter of bacterial solution. Alternatively, an oxidizing substance is added. The oxidizing substance is H2O2, and the amount added is 0.01%-0.05% of the volume of the bacterial solution.

2. The method of claim 1, wherein the liquid active bacteria preservation method of Desulfobacterium, characterized in that, In S10, the fermentation medium comprises: 0.5-1 g / L NH4Cl, 0.5-1.5 g / L MgSO4·7H2O, 3-5 g / L KH2PO4, 8-12 g / L Na2S2O3·5H2O, 3-6 g / L KNO3, 1-3 g / L NaHCO3, 0.005-0.015 g / L FeSO4·7H2O, and 0.5-1.5 mL / L trace elements, and p H is 6.5-8.0; the trace elements include EDTA-2Na at 50 g / L, CaCl2·2H2O at 7.34 g / L, FeSO4·7H2O at 5.0 g / L, MnCl2·4H2O at 2.5 g / L, ZnSO4·7H2O at 2.2 g / L, (NH4)6Mo7O2·4H2O at 0.5 g / L, CaSO4·5H2O at 0.2 g / L and NaOH at 11.0 g / L.

3. The method for liquid preservation of *Thiobacillus denitrificationus* according to claim 1, characterized in that, In S10, the denitrifying thiobacillus used is a sulfur autotrophic denitrifying bacterium. hiobacillus denitrificans SZG-SAD-004, with accession number CCTCCNO: M2023992, was obtained by inoculating 10% of the strain with two replacement treatments. Both replacement treatments removed metabolites generated during fermentation to avoid inhibiting the activity of *Thiobacillus denitrificationus* and ensure the sulfur autotrophic denitrification rate of the original bacterial culture was stably maintained at 500 mg N·L⁻¹. -1 ・h -1 above.

4. The method for liquid preservation of *Thiobacillus denitrificationus* according to claim 1, characterized in that, In step S20, the replacement ratio is 40%-100%, which is the volume ratio of the replacement culture medium to the concentrated bacterial solution; the centrifugation speed is 3000-5000 r / min, and the centrifugation time is 10-20 min; the membrane concentration uses an ultrafiltration membrane with a molecular weight cutoff of 10000-30000 Da, and the operating pressure is 0.1-0.3 MPa.

5. The method for liquid preservation of *Thiobacillus denitrificationus* according to claim 1, characterized in that, In S30, when the specific temperature is 4°C, the storage time can reach more than 90 days, and the loss rate of sulfur autotrophic denitrification rate of the bacterial solution after 90 days of storage does not exceed 30%; when the specific temperature is 28°C, the storage time is about 30 days, and the loss rate of sulfur autotrophic denitrification rate of the bacterial solution after 30 days of storage does not exceed 50%.

6. The method for liquid preservation of *Thiobacillus denitrificationus* according to claim 1, characterized in that, In step S30, the storage container is a sealed glass or plastic container with a headspace volume of 10%-20% reserved inside to balance the pressure changes of the system during storage.

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