A preservation method of nitrite oxidizing bacteria

Abstract

The present application relates to the field of microbial engineering and environmental biotechnology, and proposes a preservation method of nitrite oxidoreducing bacteria, comprising the following steps: S1, adding a nickel-containing solution into the nitrite oxidoreducing bacteria liquid, the nickel ions combine with the oxidoreductase in NOB and inhibit the enzyme activity, and the strain enters a low metabolic state; S2, preserving the bacteria liquid treated in step S1 in a 25-28 DEG C light-proof environment; S3, after the preservation is completed, removing the nickel ions in the bacteria liquid by using a chelating agent, and restoring the activity of the bacteria body. The method of the present application controls the reversible combination between the metal ions and the enzyme activity center, temporarily inhibits the metabolism of the NOB bacteria body, prolongs the preservation time of the bacteria liquid under the premise of avoiding refrigeration conditions and high-cost additives, and restores the activity of the bacteria body by removing the nickel ions before use.

Description

Technical Field

[0001] This invention relates to the fields of microbial engineering and environmental biotechnology, and in particular to a method for preserving nitrite redox bacteria. Background Technology

[0002] Nitrite-oxidizing bacteria (NOB) are a class of bacteria that can oxidize nitrite (NO2). - ) is oxidized to nitrate (NO3) - Chemoautotrophic bacteria play a crucial role in biological denitrification and are widely used in aquaculture, municipal wastewater treatment, biochemical denitrification, and agricultural drainage systems. Representative species include Nitrobacter and Nitrospira. These bacteria are extremely sensitive to environmental factors, especially to temperature changes, metal ion concentrations, and dissolved oxygen fluctuations.

[0003] In practical applications, NOB inoculants are often added in liquid or biofilm carrier form; however, they are easily inactivated or denatured under non-refrigerated conditions. Current common preservation methods, such as freeze-drying, cryopreservation, or the addition of preservatives (e.g., glycerol), have the following problems: strong reliance on refrigeration, resulting in high transportation and storage costs; drying or preservative treatment can cause irreversible damage to the bacterial membrane structure; and slow metabolic consumption continues during storage, leading to decreased activity. Furthermore, traditional preservation methods have failed to effectively address the issue of enzyme activity control. Key oxidoreductases in NOB, such as nitrite oxidoreductase (NXR), maintain a certain level of activity at room temperature, leading to continuous bacterial metabolism, energy depletion, and eventual cell death. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preserving nitrite redox bacteria, which achieves bacterial agent preservation and stable storage by reversibly regulating the activity of redox enzymes with nickel ions.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a method for preserving nitrite-oxidizing and reducing bacteria, comprising the following steps:

[0006] S1, Adding a nickel-containing solution to the nitrite redox bacteria culture causes nickel ions to bind with the redox enzyme in NOB and inhibit enzyme activity, causing the bacteria to enter a low metabolic state.

[0007] S2, the bacterial culture treated in step S1 is stored at 25-28℃ in a dark environment;

[0008] S3. After preservation, a chelating agent is used to remove nickel ions from the bacterial solution and restore bacterial activity.

[0009] Based on the above technical solutions, the preferred chelating agent is one of L-tryptophan, citric acid, and EDTA.

[0010] Based on the above technical solutions, preferably, the final concentration of L-tryptophan is 0.5-1 mM, the final concentration of citric acid is 10-15 mM, and the final concentration of EDTA is 0.6-1 mM.

[0011] Based on the above technical solutions, preferably, the nickel-containing solution is an aqueous solution of nickel chloride with a final concentration of 15-75 μM.

[0012] Based on the above technical solution, preferably, in step S1, the nitrite oxidation-reduction bacteria solution is adjusted to 800 mg NO2-N / L / h, the pH is 7-8, and the substrate NO2-N is added to a final concentration of 200 mg / L.

[0013] Based on the above technical solutions, preferably, in step S3, before the bacterial cells recover, the nickel-containing supernatant is removed by centrifugation, and then the precipitate is resuspended in a buffer solution; then a chelating agent is added, and the mixture is allowed to stand at 25-28℃ in the dark for 0.5 h to relieve the inhibitory effect of nickel ions on oxidoreductase.

[0014] Based on the above technical solutions, preferably, in step S3, the centrifugation rate is 7000-8000 rpm and the time is 10-15 min.

[0015] Based on the above technical solution, preferably, in step S3, the components of the buffer solution are NaCl 0.25-0.35 g / L, MgSO4·7H2O 0.025-0.035 g / L, FeSO4·7H2O 0.02-0.04 g / L, NaH2PO4·2H2O 0.02-0.03 g / L, and K2HPO4·3H2O 3-3.5 g / L.

[0016] Based on the above technical solutions, the preferred method is to add substrate NO2-N to 100 mg / L after the bacterial culture is restored, and then incubate at 25-28℃ for 4-6 hours. The survival rate of the bacterial culture is detected by the nitrite oxidation rate.

[0017] On the other hand, the present invention also provides the application of the above-mentioned preservation method in the transportation and preservation of nitrite redox bacteria.

[0018] Compared with existing preservation techniques for nitrite oxidoreductase (NOB) (such as refrigeration, freeze-drying, or the addition of preservatives), this invention introduces a reversible inhibition mechanism of NOB oxidoreductase by nickel ions, which has the following significant advantages in terms of technical principle, operation method, and preservation effect:

[0019] (1) Room temperature storage simplifies storage conditions. The present invention can store NOB bacterial solution at room temperature (25±2°C) without the need for complex equipment such as low temperature refrigeration, drying treatment or liquid nitrogen storage; significantly reducing energy consumption and cost in the storage and transportation process, and improving the convenience and scalability of bacterial agent application.

[0020] (2) Reversible regulation, preserving bacterial cell activity. Nickel ions reversibly inhibit the activity of oxidoreductases in NOB, causing the bacteria to enter a metabolic dormant state; before use, the inhibition of nickel ions can be relieved by simple chelation, thereby restoring the metabolic activity of the bacteria, which is superior to most cryopreservation methods.

[0021] (3) Extend the storage time and improve stability. The experimental results showed that the cell inactivation rate of NOB bacterial solution treated with nickel ions was significantly reduced during the 15-day storage period at room temperature; compared with the untreated control group, the nitrite oxidation rate could be maintained at more than 50% of the original activity.

[0022] (4) The process is simple and easy to scale up. The whole process only involves the addition of nickel salt, short-term incubation and room temperature sealed storage, without the need for complicated equipment or special materials; it is suitable for the batch preparation, transportation and temporary storage of NOB bacteria before on-site use, which is convenient for industrial promotion. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Image showing the preservation effect of bacterial culture;

[0025] Figure 2 Figure 1 shows the effect of different concentrations of nickel ions on the preservation of NOB bacterial culture at room temperature.

[0026] Figure 3 This is a diagram showing the results of a shake-flask experiment.

[0027] Figure 4 This is a diagram showing the results of an experiment in a fish tank. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Ni concentrations at different levels 2+ Evaluation of the impact of NOB bacterial culture on its activity during room temperature preservation

[0030] The NOB bacterial solution (mainly containing Nitrobacter spp.) after production in the factory was taken, and the nitrite oxidation rate of the initial bacterial solution was measured. The rate was then adjusted by dilution or concentration to stabilize it at approximately 800 mg NO2. - -N / L / h. Adjust the pH of the bacterial culture to 7.5 and add substrate NO2. - -N was added to a final concentration of 200 mg / L. The bacterial suspension was aliquoted into 500 mL polyethylene bottles, and nickel chloride hexahydrate (NiCl2·6H2O) was added to achieve final concentrations of 0 μM (control group), 15 μM, 25 μM, 50 μM, 75 μM, and 100 μM, respectively. All samples were sealed and stored in a 28°C incubator in the dark for 15 days.

[0031] Samples were taken every 3 days. After 15 days of storage, the following treatment and activity assays were performed: The bacterial culture was centrifuged at 8000 rpm for 10 min, and the nickel-containing supernatant was removed. The culture was then resuspended in NOB rate test medium (buffer: NaCl 0.3 g / L, MgSO4·7H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, NaH2PO4·2H2O 0.0265 g / L, K2HPO4·3H2O 3.27 g / L). L-tryptophan was added to a final concentration of 0.5 mM (no L-tryptophan was added to the control group), and the culture was allowed to stand for 30 minutes to chelate free Ni. 2+ To relieve enzyme inhibition; supplement with substrate NO2 - -N to 100 mg / L, incubated at 28°C and 200 rpm in the dark for 6 hours, and the nitrite oxidation rate (NO2) was measured. - (Conversion rate). Results are shown in [link to results]. Figure 2 .

[0032] Figure 2 As shown: Control group (0 μM Ni) 2+ Without the addition of regulators, the activity of NOB bacterial culture decreases rapidly under normal temperature storage conditions; adding 15-75 μM Ni... 2+The activity of the treated group decreased relatively slowly throughout the preservation period, with the 50 μM Ni group showing the most significant decrease. 2+ The treated group exhibited the best stability and recovery ability, with both low inhibitory toxicity and good reversibility; 100 μM Ni was added. 2+ The decrease was more significant in the treated group; after 15 days of storage, NO2... - The conversion rate dropped to 25%.

[0033] Example 2: Effects of different concentrations of L-tryptophan on 25 μM Ni 2+ Evaluation of the recovery effect of NOB bacterial culture activity after treatment

[0034] To study the effect of L-tryptophan on nickel ions (Ni 2+ The dose-dependent recovery of NOB bacterial activity under inhibition was investigated using the following experiment. This experiment did not involve the storage period and only examined the effect of Ni. 2+ The effect of treatment on enzyme activity inhibition and L-tryptophan relief.

[0035] Experimental design: NOB bacterial suspension produced in the factory (mainly...) was selected. Nitrobacter (Genus), after concentration or dilution adjustment, its original nitrite oxidation rate is approximately 800 mg NO2. - -N / L / h, pH controlled at 7.5. The bacterial culture was dispensed into 500 mL polyethylene bottles, and nickel chloride hexahydrate (NiCl2·6H2O) was added to each bottle to a final concentration of 25 μM. The bottles were then incubated at 25 °C for 30 minutes to induce temporary and reversible inhibition of key metabolic enzymes (such as NXR).

[0036] Nitrite oxidation rate determination: The bacterial culture was centrifuged at 7000 rpm for 15 min, and the nickel-containing supernatant was removed. The culture was then resuspended in NOB rate test medium (buffer: NaCl 0.25 g / L, MgSO4·7H2O 0.035 g / L, FeSO4·7H2O 0.02 g / L, NaH2PO4·2H2O 0.03 g / L, K2HPO4·3H2O 3 g / L). L-tryptophan was added to each culture at final concentrations of 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1.0 mM, and 2.0 mM (no L-tryptophan was added to the control group). The cultures were incubated at 25°C in the dark for 30 minutes to chelate free Ni. 2+ To relieve enzyme inhibition; take 150 mL of bacterial culture from each group and add substrate NO2 to a shake flask. - -N to 100 mg / L, incubated at 25°C and 200 rpm in the dark for 6 hours, and the nitrite oxidation rate (NO2) of each group was measured. - (conversion rate), based on the activity of the untreated original bacterial culture (800 mg NO2) -The activity recovery rate was calculated using N / L / h. The results are shown in Table 1.

[0037] Table 1. Effects of different concentrations of L-tryptophan on 25 μM Ni 2+ NOB bacterial culture activity recovery effect after treatment

[0038]

[0039] Table 1 shows that L-tryptophan can effectively chelate and deactivate Ni. 2+ The inhibitory effect on the NOB bacterial broth enzyme system was reversible, and the recovery effect increased with increasing concentration, reaching saturation at ≥0.5 mM. It is recommended to use L-tryptophan at a final concentration of 0.5 mM to ensure sufficient enzyme activity recovery while avoiding excessive addition that could cause system disturbance. This experiment further verified the effect of Ni... 2+ By regulating the controllability and reversibility of the preservation mechanism, the practicality and stability of the process are improved.

[0040] Example 3: Effects of different concentrations of citric acid on 25 μM Ni 2+ Evaluation of the recovery effect of NOB bacterial culture activity after treatment

[0041] To study the effect of citric acid on nickel ions (Ni 2+ The dose-dependent recovery of NOB bacterial activity under inhibition was investigated using the following experiment. This experiment did not involve the storage period and only examined the effect of Ni. 2+ The effect of treatment on enzyme activity inhibition and L-tryptophan relief.

[0042] Experimental design: NOB bacterial suspension produced in the factory (mainly...) was selected. Nitrobacter (Genus), after concentration or dilution adjustment, its original nitrite oxidation rate is approximately 800 mg NO2. - -N / L / h, pH controlled at 7. Dispense the bacterial culture into 500 mL polyethylene bottles, add nickel chloride hexahydrate (NiCl2·6H2O) to a final concentration of 25 μM, and let stand at 28℃ for 30 minutes to induce temporary reversible inhibition of key metabolic enzymes (such as NXR).

[0043] Nitrite oxidation rate determination: The bacterial culture was centrifuged at 7500 rpm for 12 min, and the nickel-containing supernatant was removed. The culture was then resuspended in NOB rate test medium (buffer: NaCl 0.35 g / L, MgSO4·7H2O 0.025 g / L, FeSO4·7H2O 0.04 g / L, NaH2PO4·2H2O 0.02 g / L, K2HPO4·3H2O 3.5 g / L). Citric acid was added to each culture at final concentrations of 0 mM, 2 mM, 6 mM, 10 mM, and 15 mM (no citric acid was added to the control group). The cultures were incubated at 28°C in the dark for 30 minutes to chelate free Ni.2+ To relieve enzyme inhibition; take 150 mL of bacterial culture from each group and add substrate NO2 to a shake flask. - -N to 100 mg / L, incubated at 28°C and 200 rpm in the dark for 4 hours, and the nitrite oxidation rate (NO2) of each group was measured. - (conversion rate), based on the activity of the untreated original bacterial culture (800 mg NO2) - The activity recovery rate was calculated using N / L / h. The results are shown in Table 2.

[0044] Table 2. Effects of different concentrations of citric acid on 25 μM Ni 2+ NOB bacterial culture activity recovery effect after treatment

[0045]

[0046] Table 2 shows that citric acid can complex residual Ni. 2+ The inhibition of nitrite oxidase in NOB bacterial culture was relieved. Experimental results showed that the activity recovery rate increased with increasing citric acid concentration, plateauing at 10–15 mM, with a recovery rate of approximately 67%. Although the relieving effect was slightly lower than that of L-tryptophan (72% recovery rate in Example 2), citric acid, as an inexpensive and readily available natural chelating agent, has good practical potential and is suitable for the rapid recovery of Ni²⁺-treated NOB bacteria in specific scenarios.

[0047] Example 4: Effects of different concentrations of EDTA on 25 μM Ni 2+ Evaluation of the recovery effect of NOB bacterial culture activity after treatment

[0048] To study the effect of EDTA on nickel ions (Ni 2+ The dose-dependent recovery of NOB bacterial activity under inhibition was investigated using the following experiment. This experiment did not involve the storage period and only examined the effect of Ni. 2+ The effect of treatment on enzyme activity inhibition and L-tryptophan relief.

[0049] Experimental design: NOB bacterial suspension produced in the factory (mainly...) was selected. Nitrobacter (Genus), after concentration or dilution adjustment, its original nitrite oxidation rate is approximately 800 mg NO2. - -N / L / h, pH controlled at 8. Dispense the bacterial culture into 500 mL polyethylene bottles, add nickel chloride hexahydrate (NiCl2·6H2O) to a final concentration of 25 μM, and let stand at 26℃ for 30 minutes to induce temporary reversible inhibition of key metabolic enzymes (such as NXR).

[0050] Nitrite oxidation rate determination: The bacterial culture was centrifuged at 8500 rpm for 10 min, and the nickel-containing supernatant was removed. The culture was then resuspended in NOB rate test medium (buffer: NaCl 0.35 g / L, MgSO4·7H2O 0.025 g / L, FeSO4·7H2O 0.04 g / L, NaH2PO4·2H2O 0.02 g / L, K2HPO4·3H2O 3.5 g / L). EDTA was added to each culture at final concentrations of 0 mM, 0.3 mM, 0.6 mM, 1 mM, and 1.5 mM (no EDTA was added to the control group), and the cultures were incubated at 26°C in the dark for 30 minutes to chelate free Ni. 2+ To relieve enzyme inhibition; take 150 mL of bacterial culture from each group and add substrate NO2 to a shake flask. - -N to 100 mg / L were incubated at 26°C, pH 7.5, and 200 rpm in the dark for 5 hours. The nitrite oxidation rate (NO2) of each group was then measured. - (conversion rate), based on the activity of the untreated original bacterial culture (800 mg NO2) - The activity recovery rate was calculated using N / L / h. The results are shown in Table 3.

[0051] Table 3. Effects of different concentrations of EDTA on 25 μM Ni 2+ NOB bacterial culture activity recovery effect after treatment

[0052]

[0053] Table 3 shows that EDTA, as a broad-spectrum complexing agent, can effectively chelate free Ni. 2+ Within the concentration range of 0.3–1.0 mM, EDTA can relieve the inhibition of NOB cell metabolic enzymes. Experiments show that higher EDTA concentrations do not always lead to stronger recovery effects; concentrations tend to plateau above 1.0 mM and may disturb the cell membrane system. A safe and effective concentration range of 0.6–1.0 mM EDTA is recommended, achieving over 50% activity recovery without significantly damaging the microbial structure. Although its relieving effect is not as strong as L-tryptophan or citric acid, EDTA, as a conventional chemical chelating agent, still has certain application value in specific scenarios.

[0054] Example 5: Application verification of NOB bacterial culture after preservation and revival in simulated wastewater

[0055] Based on Example 1, 25 μM Ni was selected. 2+ The bacterial culture in the treatment group was stored for 15 days and then treated with L-tryptophan to remove inhibition, serving as an active bacterial agent for simulated wastewater degradation experiments.

[0056] Experimental design: In each 150 mL shake flask system, 100 mg / L NO2 was added. - -N and 2 mg / LPO4 3- Simulated wastewater was used as the reaction substrate. Subsequently, NOB bacterial culture with restored activity after treatment was added at a ratio of 5% (v / v). Each group of shake flasks was placed in a shaker at 30°C and 200 rpm under light-protected conditions for shaking incubation. The entire reaction process lasted at least 24 hours, with samples collected every 6 hours to detect nitrite (NO2). - -N) and nitrates (NO3) - The concentration change of (-N) was used to evaluate the nitrite oxidation capacity of the NOB inoculant after nickel ion treatment and revival. Results are shown below. Figure 3 .

[0057] Figure 3 As shown: after treatment with 25 μM Ni 2+ The bacterial culture, revived after 15 days of preservation, still exhibited good nitrite oxidation function, reducing NO2 levels within 24 hours. - When the -N concentration drops below 5 mg / L, NO3... - -N formation reached over 95 mg / L, NO2 - -N is almost completely converted into NO3. - -N indicates that the bacterial solution preserved by the method of the present invention still has high activity in wastewater treatment scenarios and can be directly used for saline wastewater treatment.

[0058] Example 6: Application verification of NOB bacterial solution after preservation and revival in aquarium environment

[0059] Similarly, the 25 μM Ni from Example 1 was selected. 2+ The bacterial culture in the treatment group was preserved for 15 days and then revived by L-tryptophan treatment to verify its effect on removing low concentrations of nitrite in aquarium water.

[0060] Experimental Design: The experiment was conducted in 20 L glass aquariums, with 5 goldfish stocked in each tank to simulate a typical aquarium environment. Nitrite was added to the water before the experiment to initially reduce NO2 levels. - The NO-N concentration was set at approximately 0.5 mg / L to simulate a slightly saline environment in aquarium water. Subsequently, NOB-active bacteria solution, which had been treated with nickel ions and stored at room temperature for 15 days and then revived, was added at a ratio of 1‰ of the water volume. The fish in the aquarium were fed a fixed amount daily to ensure a continuous production of nitrogenous metabolites. For the next 7 days, samples were taken daily at regular intervals to test the NO2 concentration in the water. - -N and NO3 -Changes in -N concentration were observed, along with the health status of the fish, to comprehensively evaluate the actual performance of the administered microbial agent in removing nitrite and its ecological adaptability in the aquarium environment. Results are shown below. Figure 4 .

[0061] Figure 4 As shown: via Ni 2+ NOB bacterial solution, after being treated and stored for 15 days, can quickly initiate the nitrite oxidation reaction in aquarium applications after revival, thus reducing NO2. - The nitrification system was stabilized and reduced to a safe concentration (<0.2 mg / L) and maintained stable operation. The water body remained stable throughout the process, and the fish showed good vitality and no stress response, indicating that it has good practical application value and ecological compatibility.

[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 within the protection scope of the present invention.

Claims

1. A method for preserving nitrite-oxidizing and reducing bacteria, characterized in that, Includes the following steps: S1, a nickel-containing solution is added to the nitrite redox bacteria culture. The nickel ions bind to the redox enzyme in NOB and inhibit the enzyme activity, causing the bacteria to enter a low metabolic state. The nickel-containing solution is an aqueous solution of nickel chloride with a final concentration of 15-75 μM. S2, the bacterial culture treated in step S1 is stored at 25-28℃ in a dark environment; S3. After preservation, a chelating agent is used to remove nickel ions from the bacterial solution and restore bacterial activity. The chelating agent is one of L-tryptophan, citric acid, and EDTA; the final concentration of L-tryptophan is 0.5-1 mM, the final concentration of citric acid is 10-15 mM, and the final concentration of EDTA is 0.6-1 mM.

2. The method for preserving nitrite-oxidizing and reducing bacteria as described in claim 1, characterized in that, In step S1, the nitrite oxidation-reduction bacteria culture solution is adjusted to 800 mg NO2-N / L / h, pH is 7-8, and substrate NO2-N is added to a final concentration of 200 mg / L.

3. The method for preserving nitrite-oxidizing and reducing bacteria as described in claim 1, characterized in that, In step S3, before the cells recover, the nickel-containing supernatant is removed by centrifugation, and then the precipitate is resuspended in buffer solution. Then, a chelating agent is added, and the mixture is allowed to stand at 25-28℃ in the dark for 0.5 h to relieve the inhibitory effect of nickel ions on oxidoreductase.

4. The method for preserving nitrite-oxidizing and reducing bacteria as described in claim 3, characterized in that, In step S3, the centrifugation rate is 7000-8000 rpm and the time is 10-15 min.

5. The method for preserving nitrite-oxidizing and reducing bacteria as described in claim 3, characterized in that, In step S3, the buffer solution consists of NaCl 0.25-0.35 g / L, MgSO4·7H2O 0.025-0.035 g / L, FeSO4·7H2O 0.02-0.04 g / L, NaH2PO4·2H2O 0.02-0.03 g / L, and K2HPO4·3H2O 3-3.5 g / L.

6. The method for preserving nitrite-oxidizing and reducing bacteria as described in claim 1, characterized in that, After the bacterial culture was restored, the substrate NO2-N was added to 100 mg / L, and then the culture was incubated at 25-28℃ for 4-6 h. The survival rate of the bacterial culture was detected by the nitrite oxidation rate.

7. The application of the preservation method according to any one of claims 1-6 in the transportation and preservation of nitrite redox bacteria.

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