Post-treatment liquid of fermentation nitrifying bacterial agent and preservation method

Abstract

The invention relates to the technical field of microbial inoculum preservation, and provides a post-treatment liquid of a fermentation nitrifying microbial inoculum and a preservation method, the post-treatment liquid comprises inorganic salt, trace elements and a protective agent, and the protective agent is cellulose, xanthan gum and sodium bentonite. The cellulose, the xanthan gum and the sodium bentonite have a synergistic effect to form a dynamic balance system. The cellulose and the xanthan gum are subjected to physical action, and the bentonite is subjected to chemical exchange action, so that the acid-base fluctuation is alleviated on the whole, and the pH value is kept in a proper range, thereby ensuring the stability of the fermentation nitrifying bacterial agent in the storage period and the efficient operation of the nitrifying process.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial agent preservation, in particular to a post-processing liquid of a fermentation nitrifying bacterial agent and a preservation method. Background Art

[0002] The principle of microbial preservation is to artificially create conditions that keep microorganisms in a dormant state, such as low temperature and dryness, based on their physiological and biochemical characteristics. A good preservation method must not only maintain the activity of the strain over a long period of time but also be simple and economical to facilitate widespread application in production.

[0003] Existing preservation methods use complex nitrifying agent treatment processes, fail to adopt industry standards to detect ammonia oxidation rate losses, or require co-cultivation for short-term room-temperature storage. For example, the preservation method for nitrifying bacteria disclosed in CN 106554921A describes nitrifying bacteria derived from intermittent activated sludge, making it suitable for frozen strain preservation. For example, the dry preservation method for nitrifying bacteria disclosed in CN 113122454A involves the addition of protective agents, vacuum drying, and mixing with resting cells, making it difficult to apply industrially. Both of these preservation methods require a low-temperature storage environment and have stringent temperature requirements. CN 114686392A discloses a method for preserving nitrifying bacteria at room temperature, wherein the method involves co-culturing nitrifying bacteria and microalgae at room temperature. This method requires alternating light and dark cultivation, and the total daylight exposure time is greater than 8 hours. Due to the limited light, the preservation environment is also relatively harsh. Therefore, a method for preserving nitrifying bacteria fermentation broth at room temperature that can maintain high bacterial activity is needed. Summary of the Invention

[0004] In view of this, the present invention proposes a post-treatment liquid and a preservation method for a fermentation nitrifying bacteria agent that can be preserved at room temperature and maintain high activity of the bacteria.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a post-treatment liquid of a fermentation nitrifying bacteria agent, wherein the post-treatment liquid comprises inorganic salts, trace elements and a protective agent, and the protective agent comprises cellulose, xanthan gum and sodium bentonite.

[0006] The cellulose network provides a stable framework for the entire suspension. This physical support helps maintain uniform bacterial distribution, reducing the risk of localized pH fluctuations leading to excessive or insufficient pH. It also serves as a carbon source to maintain bacterial activity. Xanthan gum improves the rheological properties of the post-treatment fluid, facilitating uniform bacterial dispersion and preventing aggregation. Sodium bentonite absorbs metabolic products, maintaining pH stability and reducing NO₃ loss. Cellulose, xanthan gum, and sodium bentonite work synergistically to form a dynamic equilibrium system. The physical interaction of cellulose and xanthan gum, and the chemical exchange of bentonite, mitigate overall pH fluctuations and maintain a suitable pH range.

[0007] A stable pH is crucial for the activity of nitrifying bacteria. When the pH is unstable, the nitrification process is inhibited or even reversed, leading to the loss of NO3. Maintaining a stable pH ensures efficient nitrification. Stable operating conditions (such as an appropriate pH) enable nitrifying bacteria to more efficiently convert ammonia nitrogen into more easily removable nitrates, thereby improving wastewater treatment efficiency. At the same time, reducing the loss of nutrients and the accumulation of degradation byproducts further enhances the nitrifying bacteria's ability to treat pollutants.

[0008] On the basis of the above technical solution, preferably, the mass ratio of cellulose:xanthan gum:sodium bentonite is 20-25:10-15:3-4.

[0009] Based on the above technical solution, preferably, the components of the inorganic salt are: NaCl 0.3-0.5 g / L, MgSO4·7H2O 0.03-0.05 g / L, K2HPO4·3H2O 0.3-0.5 g / L, and KH2PO4 0.01-0.05 g / L.

[0010] Based on the above technical solution, preferably, the components of the trace elements are: 10-20 g / L MnCl2·4H2O, 15-20 g / L Na2B4O7·10H2O, 15-25 g / L ZnSO4·7H2O, 8-15 g / L EDTA-Fe, 5-8 g / L CoSO4·7H2O, 4-6 g / L CuSO4·5H2O and 3-8 g / L NiSO4·6H2O.

[0011] Iron is a crucial component for electron transfer and enzymatic reactions in nitrifying bacteria. Chelated iron (EDTA-Fe) not only provides a sufficient iron source but also prevents iron precipitation in solution, ensuring its effective bioavailability. CoSO4·7H2O, CuSO4·5H2O, and NiSO4·6H2O help enhance enzyme catalytic activity, further improving the efficiency of the nitrification process.

[0012] By providing a stable physical and chemical environment, the protective agent ensures the long-term and stable presence of trace elements, preventing element loss or biological unavailability due to changes in the external environment. At the same time, trace elements enhance the physiological activity of the bacteria and, in synergy with the protective agent, create a microenvironment conducive to bacterial growth and metabolism. By improving the overall stability of the liquid and providing the physiological foundation provided by the trace elements, the post-treatment liquid maintains a highly effective protective effect over a wide temperature range (4°C to 28°C), ensuring the long-term retention of the activity and function of nitrifying bacteria.

[0013] On the basis of the above technical solution, preferably, the dosage of the trace elements is 8-15 ml / L, and the dosage of the protective agent is 2-6 g / L.

[0014] On the basis of the above technical solution, preferably, the cellulose is carboxymethyl cellulose or hydroxyethyl cellulose.

[0015] On the other hand, the present invention also provides a method for preserving a fermentation nitrifying bacteria agent, comprising mixing a post-treatment liquid and a fermentation nitrifying bacteria agent, adjusting the pH of the mixture to 7.0-7.5, and preserving the mixture at 4°C-28°C.

[0016] On the basis of the above technical solution, preferably, the volume ratio of the post-treatment liquid: fermentation nitrifying bacteria agent is 1: (1-1.5).

[0017] On the basis of the above technical solution, preferably, the fermentation nitrifying bacteria agent is a bacterial agent obtained by co-fermentation of nitrite bacteria and nitrifying bacteria.

[0018] The post-treatment liquid and preservation method of a fermentation nitrifying bacteria agent of the present invention have the following beneficial effects compared with the prior art: the post-treatment liquid of the present invention can stabilize the pH during storage, reduce the loss of NO3 in the nitrifying bacteria agent, and can be preserved at 4-28°C, while also improving the application effect of the nitrifying bacteria agent in landfill leachate and aniline wastewater. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] The fermentation nitrifying bacteria agent of the present invention is a bacterial agent obtained by co-fermentation of nitrosobacteria and nitrifying bacteria, wherein the nitrosobacteria include Nitrosomonas europaea AT7 and Nitrosomonas europaea C-31, and the two bacteria are mixed in a ratio of 1:1. The nitrifying bacteria is Nitrobacter Winogradskyi Y3-2.

[0021] The nitrosating bacteria tank is connected to the nitrifying bacteria tank. The fermentation method is as follows: the nitrosating bacteria tank is inoculated with nitrosating bacteria at 10% (v / v), sodium carbonate solution is added during the culture process to adjust the pH to 7.5, the temperature is controlled at 30°C, aeration is performed intermittently, the dissolved oxygen is controlled at 5mg / L, and the culture time is 5-7 days. After the ammonia nitrogen in the culture medium is consumed, the cultured nitrosating bacteria are obtained, the bacteria are allowed to stand and separate, and the supernatant overflows into the nitrifying bacteria tank; the nitrifying bacteria tank is inoculated with nitrifying bacteria at 10% (v / v), sodium carbonate solution is added during the culture process to adjust the pH to 7.5, the temperature is controlled at 30°C, aeration is performed intermittently, the dissolved oxygen is controlled at 5mg / L, and the culture time is 5-7 days. After the nitrite in the culture medium is consumed, the cultured nitrifying bacteria are obtained. After the fermentation is completed, the nitrosating bacteria and the nitrifying bacteria are respectively transported to storage tanks via pipelines.

[0022] The chemical reagents used in the present invention were all purchased from the market, among which sodium bentonite was purchased from Hunan Xianghuan New Materials Co., Ltd.

[0023] Example 1

[0024] This embodiment provides a method for preserving a fermentation nitrifying bacteria agent, comprising the following steps:

[0025] S1, prepare a post-treatment liquid formula, the components of the post-treatment liquid are inorganic salts, trace elements and protective agents, and the protective agents are carboxymethyl cellulose, xanthan gum and sodium bentonite.

[0026] The components of inorganic salts are: NaCl 0.4g / L, MgSO4·7H2O 0.04g / L, K2HPO4·3H2O 0.4g / L, KH2PO40.03g / L, trace elements 10ml / L, and protective agent 4g / L.

[0027] The trace element composition is: 16g / L MnCl2·4H2O, 18g / L Na2B4O7·10H2O, 22g / L ZnSO4·7H2O, 12g / L EDTA-Fe, 7g / L CoSO4·7H2O, 5g / L CuSO4·5H2O and 6g / L NiSO4·6H2O.

[0028] The mass ratio of carboxymethyl cellulose: xanthan gum: sodium bentonite is 22:13:4, and 1 L of post-treatment liquid is prepared according to the components and ratio.

[0029] S2. Mix the post-treatment liquid and the fermentation nitrifying bacteria agent in a volume ratio of 1:1, adjust the pH of the mixture to 7.3, and store it at 4°C, 14°C, and 28°C for 90 days. Calculate the changes in ammonia oxidation rate, pH value, and NO3 during storage. The detection method refers to the "Performance Evaluation Method of Nitrifying Bacteria for Water Treatment".

[0030] Compared with Example 1, the CK1 group did not add a protective agent, the CK2 group lacked sodium bentonite compared with Example 1, the CK3 group lacked xanthan gum compared with Example 1, and the CK4 group did not add trace elements. The storage temperature of CK1 to 4 was all set at 14°C.

[0031] Table 1 Ammonia oxidation rate of nitrifying bacteria at different storage temperatures

[0032]

[0033] As shown in Table 1, after adding a protective agent, the fermented nitrifying bacteria inoculum maintained an ammonia oxidation rate exceeding 90% after storage at 4-28°C for 90 days. The ammonia oxidation rate was significantly reduced in the groups without a protective agent, or when the protective agent lacked sodium bentonite or xanthan gum, as well as when no trace elements were added. This demonstrates that both the protective agent and the trace elements can contribute to maintaining the ammonia oxidation rate of the inoculum.

[0034] Table 2 pH changes of nitrifying bacteria during storage

[0035] Grouping 0d 30d 45d 60d 90d 4℃ 7.3 7.31 7.23 7.25 7.41 14℃ 7.3 7.33 7.21 7.22 7.4 28℃ 7.3 7.35 7.29 7.32 7.45 CK1 7.3 7.11 8.23 8.34 8.45 CK2 7.3 7.64 7.56 7.63 7.95 CK3 7.3 7.66 7.57 7.67 8.03 CK4 7.3 7.4 7.51 7.38 7.94

[0036] As shown in Table 2, the addition of a preservative stabilized the pH of the inoculum during storage, preventing pH fluctuations and maintaining high activity. Groups without a preservative, or without sodium bentonite or xanthan gum, or without trace elements, experienced significant pH fluctuations, leading to decreased activity (see Table 1). This suggests that both preservatives and trace elements can maintain the pH of the inoculum, preventing significant pH fluctuations during storage that could affect bacterial activity.

[0037] Table 3 Changes of NO3 during storage

[0038]

[0039] As shown in Table 2, after adding the protective agent, the NO3 content of the inoculant was maintained at over 90% during storage. In the groups without the protective agent, or with the protective agent lacking sodium bentonite or xanthan gum, and without the addition of trace elements, the NO3 content of the inoculant decreased significantly, reaching only 51% to 69% at 90 days. This indicates that the protective agent and trace elements can maintain the NO3 content of the inoculant and keep the nitrification process efficient.

[0040] Example 2

[0041] A post-treatment liquid of a fermentation nitrifying bacteria agent comprises inorganic salts, trace elements and a protective agent, wherein the protective agent is hydroxyethyl cellulose, xanthan gum and sodium bentonite.

[0042] The components of inorganic salts are: NaCl 0.3g / L, MgSO4·7H2O 0.05g / L, K2HPO4·3H2O 0.3g / L, KH2PO40.05g / L, trace elements 8ml / L, and protective agent 6g / L.

[0043] The trace element composition is: 10g / L MnCl2·4H2O, 20g / L Na2B4O7·10H2O, 15g / L ZnSO4·7H2O, 15g / L EDTA-Fe, 5g / L CoSO4·7H2O, 6g / L CuSO4·5H2O and 3g / L NiSO4·6H2O.

[0044] The mass ratio of hydroxyethyl cellulose: xanthan gum: sodium bentonite is 20:15:3, and 1 L of post-treatment liquid is prepared according to the components and proportions.

[0045] S2, mixing the post-treatment liquid and the fermentation nitrifying bacteria agent in a volume ratio of 1:1.2, adjusting the pH of the mixed liquid to 7.5, and storing them at 25°C.

[0046] Example 3

[0047] A post-treatment liquid of a fermentation nitrifying bacteria agent comprises inorganic salts, trace elements and a protective agent, wherein the protective agent is carboxymethyl cellulose, xanthan gum and sodium bentonite.

[0048] The components of inorganic salts are: NaCl 0.5g / L, MgSO4·7H2O 0.03g / L, K2HPO4·3H2O 0.5g / L, KH2PO40.01g / L, trace elements 15ml / L, and protective agent 2g / L.

[0049] The trace element composition is: 20g / L MnCl2·4H2O, 15g / L Na2B4O7·10H2O, 25g / L ZnSO4·7H2O, 8g / L EDTA-Fe, 8g / L CoSO4·7H2O, 4g / L CuSO4·5H2O and 8g / L NiSO4·6H2O.

[0050] The mass ratio of carboxymethyl cellulose: xanthan gum: sodium bentonite is 25:10:3.5, and 1 L of post-treatment liquid is prepared according to the components and proportions.

[0051] S2, mixing the post-treatment liquid and the fermentation nitrifying bacteria agent in a volume ratio of 1:1.5, adjusting the pH of the mixed liquid to 7.0, and storing them at 28°C.

[0052] Table 4 Changes in activity and pH during storage of Examples 1-3

[0053]

[0054] By adjusting the concentration of each component, there was no significant difference in the ammonia oxidation rate, pH value and NO3 of Examples 1-3 during storage, and Example 1 had the best effect.

[0055] The nitrifying bacteria agent treated with post-treatment liquid was added to landfill leachate, aniline wastewater and high-salt wastewater, and the changes in ammonia nitrogen concentration were detected at regular intervals.

[0056] The leachate was collected from the Chenjia Chong landfill in Wuhan and used after pre-aeration and dilution. Aniline wastewater was collected from a petrochemical company in Nanjing, with an aniline content of 3-10 mg / L. High-salt wastewater was collected from a food wastewater company in Sichuan, with a salt content of approximately 12,000 mg / L.

[0057] The initial ammonia nitrogen concentration of all wastewater was adjusted to 100-130 mg / L, and potassium dihydrogen phosphate was added in an appropriate amount to make N:P≈5:1. The pH of the wastewater was adjusted to about 8.0 with sodium carbonate solution, and the wastewater was divided into 500ml triangular flasks, 150ml per bottle, and the treated nitrifying bacteria solution was added at a dosage of 1ml / 150ml. The test results are shown in the table below.

[0058] Table 5 Wastewater treatment effect

[0059]

[0060]

[0061] As shown in Table 4, due to the addition of a protective agent in the embodiment of the present invention, the ammonia nitrogen removal rate can reach more than 95% under the same time conditions. On the contrary, no protective agent is added or the protective agent lacks certain components, and the lack of trace elements will affect the ammonia nitrogen removal efficiency. The results are the same as those in Tables 1-4, which proves that the protective agent and trace elements can not only maintain the pH value of the bacterial agent, but also maintain a high activity and ammonia nitrogen removal rate.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A post-treatment liquid of a fermentation nitrifying bacteria agent, characterized in that: The post-treatment liquid comprises inorganic salts, trace elements and protective agents, and the protective agents comprise cellulose, xanthan gum and sodium bentonite.

2. The post-treatment liquid of a fermentation nitrifying bacteria agent according to claim 1, characterized in that: The mass ratio of the cellulose:xanthan gum:sodium bentonite is 20-25:10-15:3-4.

3. The post-treatment liquid of a fermentation nitrifying bacteria agent according to claim 1, characterized in that: The inorganic salt comprises the following components: NaCl 0.3-0.5 g / L, MgSO4·7H2O 0.03-0.05 g / L, K2HPO4·3H2O 0.3-0.5 g / L, and KH2PO4 0.01-0.05 g / L.

4. The post-treatment liquid of a fermentation nitrifying bacteria agent according to claim 1, characterized in that: The trace element components are: 10-20 g / L MnCl2·4H2O, 15-20 g / L Na2B4O7·10H2O, 15-25 g / L ZnSO4·7H2O, 8-15 g / L EDTA-Fe, 5-8 g / L CoSO4·7H2O, 4-6 g / L CuSO4·5H2O and 3-8 g / L NiSO4·6H2O.

5. The post-treatment liquid of a fermentation nitrifying bacteria agent according to claim 1, characterized in that: The dosage of the trace elements is 8-15 ml / L, and the dosage of the protective agent is 2-6 g / L.

6. The post-treatment liquid of a fermentation nitrifying bacteria agent according to claim 1, characterized in that: The cellulose is carboxymethyl cellulose or hydroxyethyl cellulose.

7. A method for preserving a fermentation nitrifying bacteria agent, characterized in that: The post-treatment liquid according to any one of claims 1 to 4 is mixed with a fermentation nitrifying bacteria agent, the pH of the mixed liquid is adjusted to 7.0 to 7.5, and the mixed liquid is stored at 4° C. to 28° C.

8. The method for preserving a fermentation nitrifying bacteria agent according to claim 7, wherein: The volume ratio of the post-treatment liquid to the fermentation nitrifying bacteria agent is 1: (1-1.5).

9. The method for preserving a fermentation nitrifying bacteria agent according to claim 7, wherein: The fermentation nitrifying bacteria agent is a bacterial agent obtained by co-fermentation of nitrite bacteria and nitrifying bacteria.

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