A high-density fermentation method for nitrite-oxidizing bacteria enrichment liquid

Abstract

The application discloses a high-density fermentation method of nitrite-oxidizing bacteria enrichment liquid, which comprises the following steps: preparing the nitrite-oxidizing bacteria enrichment liquid into a nitrite-oxidizing bacteria seed liquid, and performing low-temperature treatment on the seed liquid; connecting the seed liquid into a fermentation culture medium, and simultaneously connecting yeast into the fermentation culture medium for fermentation; supplementing low-concentration nitrite nitrogen in the early stage of the fermentation, and supplementing high-concentration nitrite nitrogen in the middle and late stages of the fermentation, and ending the fermentation when the fermentation rate no longer increases. The high-density fermentation method provided by the application effectively improves the fermentation level of the nitrite-oxidizing bacteria enrichment liquid, and weakens the influence of miscellaneous bacteria in the fermentation process, and provides a solution for the technical problem of low culture rate of the nitrite-oxidizing bacteria which cannot be cultured in a pure way.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a high-density fermentation method for nitrite-oxidizing bacterial enrichment solutions. Background Technology

[0002] Nitrification is the process by which nitrifying microorganisms convert NH4+ into nitrogenous oxygen. + Oxidized to NO3 - The nitrification process, which releases energy, can be divided into two steps: microorganisms convert NH4+ into nitrogen dioxide and nitrogen dioxide. + Oxidized to NO2 - Microorganisms will produce NO2 - Oxidized to NO3 - Furthermore, these two steps are typically accomplished by different microorganisms.

[0003] Nitrite-oxidizing bacteria (NOB) participate in the second step of the nitrification reaction, namely, NO2. - Oxidized to NO3 - In this process, NOB obtains energy and uses inorganic carbon sources such as CO2 to synthesize organic matter, making it an important type of inorganic chemoautotrophic bacteria. Among them, Nitrifying Bacillus winogradskyi is a relatively common type of nitrite-oxidizing bacteria in nature. It belongs to facultative chemoautotrophic bacteria, has a long generation time, and grows slowly.

[0004] Currently, fermentation methods for pure strains of nitrite-oxidizing bacteria have been reported in the literature. However, most nitrite-oxidizing bacteria have not yet yielded pure cultures, and only high-purity enrichment solutions can be obtained. While high-purity enrichment solutions of nitrite-oxidizing bacteria can achieve high-density fermentation, in the middle and later stages of fermentation, the high concentration of nitrates produced by the metabolism of nitrite-oxidizing bacteria and the remaining organic matter in the culture medium lead to the rapid proliferation of denitrifying bacteria, which in turn interferes with the growth and fermentation of the target bacteria (i.e., nitrite-oxidizing bacteria), resulting in the inability to obtain high-rate microbial inoculant products. Summary of the Invention

[0005] To address the problem of interference from other microorganisms in high-density fermentation of nitrite-oxidizing bacteria enriched broth, this invention provides a novel fermentation method for nitrite-oxidizing bacteria enriched broth. This method can effectively inhibit the rapid proliferation of denitrifying bacteria in the later stages of high-density fermentation, thereby improving the fermentation level of nitrite-oxidizing bacteria and obtaining high-rate microbial inoculant products.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A high-density fermentation method for nitrite-oxidizing bacteria enrichment broth includes the following steps:

[0008] S1. Prepare nitrite-oxidizing bacteria seed solution from nitrite-oxidizing bacteria enrichment solution and refrigerate the nitrite-oxidizing bacteria seed solution.

[0009] S2. The refrigerated nitrite-oxidizing bacteria seed culture is inoculated into a sterilized fermentation medium, and yeast is simultaneously inoculated for mixed fermentation. The fermentation process is divided into two stages:

[0010] In the early stage of fermentation, whenever the nitrite nitrogen in the system is consumed, nitrite nitrogen is added and its final concentration in the fermentation medium is ≤100mg / L; the early stage of fermentation as described in this invention specifically refers to when the nitrate of the fermentation product in the fermentation system is <300mg / L.

[0011] During the later stages of fermentation, whenever nitrite nitrogen in the system is depleted, nitrite nitrogen is added and its final concentration in the fermentation medium is ≥300 mg / L. Fermentation is terminated when the fermentation rate no longer increases.

[0012] Furthermore, in the above-mentioned high-density fermentation method, the nitrite-oxidizing bacterial enrichment broth has Nitrifying Bacillus vesiculosus as the dominant bacterial species, and Nitrifying Bacillus vesiculosus accounts for more than 70% of the total bacterial count of the enrichment broth.

[0013] Furthermore, in the above-mentioned high-density fermentation method, the rate of the nitrite-oxidizing bacteria seed liquid in step S1 is 110±15 mg NO2-N / L / h.

[0014] In a specific embodiment of the present invention, the seed culture of nitrite-oxidizing bacteria is prepared by the following method: the nitrite-oxidizing bacteria enrichment solution is inoculated into a sterilized fermentation medium (inoculation amount is 10-15%), nitrite nitrogen is added to make the final concentration of nitrite nitrogen in the fermentation medium reach 100 mg / L, and the culture is shaken. When the nitrite nitrogen in the fermentation medium is consumed, nitrite nitrogen is immediately added to make the final concentration of nitrite nitrogen reach 100 mg / L. Multiple batches of feeding are carried out until the seed culture collection rate is 110 ± 15 mg NO2-N / L / h.

[0015] Furthermore, in the above-mentioned high-density fermentation method, the inoculation amount of the nitrite-oxidizing bacteria seed liquid in step S2 is 10-15%, and the inoculation amount of the yeast is 0.1-0.5%.

[0016] Furthermore, the yeast used can be Candida utilis; Candida utilis has a short generation time, can quickly consume organic matter, can survive in alkaline environments, and is non-toxic to the environment.

[0017] Furthermore, in the above-mentioned high-density fermentation method, the refrigeration time is 3-15 days, with 3-7 days being optimal; the refrigeration temperature can be selected from commonly used temperatures in the field, such as 4℃.

[0018] The data from the examples show that low-temperature treatment of the seed solution can effectively improve the activity of nitrite-oxidizing bacteria in the enrichment solution.

[0019] Furthermore, in the above-mentioned high-density fermentation method, the fermentation medium contains organic matter, and the organic matter includes, but is not limited to, one or more of yeast extract, glucose, and glycerol, with glycerol being the best.

[0020] Furthermore, in the above-mentioned high-density fermentation method, the pH of the fermentation medium is 6.5-7.5, and the pH is maintained constant during the fermentation process.

[0021] In one specific embodiment of the present invention, the fermentation medium consists of: NaCl 0.3 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 0.1 g / L, organic matter 1 g / L, trace element mother liquor 1 mL / L, and carrier 1 g / L.

[0022] In the above fermentation medium, the trace element mother liquor contains one or more of FeSO4, CuSO4, CoCl2, MnSO4, ZnSO4, NiCl2, and H3BO3, and the carrier is one or more of CaCO3, mica, zeolite powder, and diatomaceous earth.

[0023] The microbial agent products prepared by the high-density fermentation method of the nitrite-oxidizing bacteria enrichment solution provided by the present invention should also fall within the protection scope of the present invention.

[0024] In the high-density fermentation method provided by this invention, *Nitrobacterium velutipes* is a facultative chemoautotrophic bacterium, meaning it can also metabolize and utilize organic matter. Therefore, adding organic matter during the lag phase when the initial bacterial concentration is low can promote its accelerated growth. However, in addition to the target bacterium (i.e., *Nitrobacterium velutipes*), the enrichment solution also contains a small amount of heterotrophic bacteria, which grow synchronously with the target bacterium. Compared to the heterotrophic bacteria, the target bacterium has a longer generation time and slower growth. Therefore, in the early stage of fermentation, the heterotrophic bacteria will rapidly multiply using the organic matter in the culture medium. Moreover, if the organic matter content in the culture medium is low, the heterotrophic bacteria will rapidly metabolize the organic matter before the target bacterium enters the logarithmic phase, resulting in no organic matter available for the target bacterium before the high-speed growth phase is reached.

[0025] Furthermore, in the later stages of fermentation, the high concentration of nitrates produced by nitrite-oxidizing bacteria and the abundance of organic matter in the fermentation medium are more conducive to the rapid proliferation of denitrifying bacteria. Moreover, data from the examples show that even without the introduction of organic matter into the fermentation medium, denitrifying bacteria can use the metabolic products of nitrite-oxidizing bacteria, or even their cells, as organic matter for growth in the later stages of fermentation. To address this issue, this invention, on the one hand, involves refrigerating the prepared seed culture to further enhance the activity of nitrite-oxidizing bacteria; on the other hand, yeast is introduced into the fermentation system. In the early stages of fermentation, the yeast assists in metabolizing the organic matter in the system, and, combined with high-speed feeding, creates a more suitable growth environment for nitrite-oxidizing bacteria. This inhibits the growth of denitrifying bacteria before the nitrate concentration in the system increases. In the later stages, a feeding-acceleration strategy is used to kill the yeast in the system using the high concentration of the substrate nitrite.

[0026] The beneficial effects of this invention are as follows: The high-density fermentation method provided by this invention effectively improves the fermentation level of nitrite-oxidizing bacteria and reduces the influence of miscellaneous bacteria during the fermentation process. Therefore, the fermented nitrite-oxidizing bacteria have a high culture rate and can quickly achieve the degradation of nitrite. Detailed Implementation

[0027] To better understand the present invention, specific embodiments are provided below to further illustrate its content. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, all examples were conducted under standard experimental conditions or as recommended in the manufacturer's instructions. All reagents and materials used are commercially available unless otherwise specified.

[0029] Example 1: Co-fermentation experiment of yeast and nitrite-oxidizing bacteria in a 5L tank.

[0030] The yeast used in this example is Candida albicans, and the nitrite-oxidizing bacterial enrichment solution uses Nitrobacter velutipes as the dominant species. The specific high-density fermentation process in this example is as follows:

[0031] (1) Preparation of NOB fermentation medium: NaCl 0.3 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 0.1 g / L, organic matter 1 g / L, trace element stock solution (containing FeSO4, CoCl2, MnSO4 and NiCl2, with each component having a 1× concentration of 0.2 mg / L) 1 mL / L, carrier (CaCO3) 1 g / L. Sterilize at 121℃ for 30 min and adjust the pH to 7.0. The liquid volume is 3 L / 5 L.

[0032] (2) Preparation and low-temperature treatment of seed culture: The nitrite oxidation enrichment solution was inoculated into sterilized NOB fermentation medium, and sodium nitrite was added to bring the final concentration of nitrite nitrogen to 100 mg / L. Whenever the nitrite nitrogen in the system was consumed, sodium nitrite was added again to bring the final concentration of nitrite nitrogen to 100 mg / L. The culture was terminated after 5 batch feedings. The seed culture collection rate was 110 ± 15 mg NO2-N / L / h. The seed culture was stored at 4℃ for 7 days.

[0033] (3) Fermentation: After inoculating the seed culture at 10%, the *Candida* culture (obtained by culturing *Candida* in YPD medium to the logarithmic growth phase) was centrifuged and resuspended in fermentation medium. This resuspension was repeated twice before inoculation into the fermentation medium at an inoculum size of 0.5%. For the first 48 hours of fermentation, a certain amount of sodium nitrite was added immediately after the previously added nitrite was consumed, bringing the final nitrite concentration in the tank to 100 mg / L. After 48 hours, another certain amount of sodium nitrite was added immediately after the previously added nitrite was consumed, bringing the final nitrite concentration in the tank to 300 mg / L. Fermentation conditions and feed amounts were consistent across all groups. Fermentation was stopped when the rate no longer increased.

[0034] Following the above process, we simultaneously established a control group without low-temperature treatment (no low-temperature refrigeration and no inoculation with Candida albicans, otherwise the process was the same as above), a low-temperature treatment control group (only no inoculation with Candida albicans, otherwise the process was the same as above), and a group without low-temperature treatment + Candida albicans (only no low-temperature treatment, otherwise the process was the same as above). The fermentation results of each group were then tested.

[0035] The test results are shown in Table 1. As can be seen from Table 1, the mixed fermentation of Candida albicans and nitrite-oxidizing bacteria inhibited the competition of other bacteria for nutrients in the later stage, which can further improve the fermentation rate of nitrite-oxidizing bacteria. Combined with the seed treatment at low temperature, the problem of not being able to speed up the later stage was solved, and the 48h-bottom fermentation rate can be increased by more than 100%.

[0036] The method for detecting the nitrite oxidation rate is as follows: Activated bacterial agent is added to the test culture medium, and samples are taken at 0h, 0.5h, 1.5h, 2.5h, and 3.5h to detect the nitrite concentration. The rate is calculated by fitting a curve of this concentration versus time. The activation operation involves taking 100mL of the bacterial agent product, adding sodium nitrite to a final concentration of 200mg NO2-N / L, and incubating at 30℃ in a shaker for at least 3h. The test culture medium consists of: NaNO2 0.5g / L, NaCl 0.3g / L, MgSO4·7H2O 0.03g / L, FeSO4·7H2O 0.03g / L, NaH2PO3·2H2O 0.265g / L, K2HPO3·3H2O 3.27g / L, pH 7.8. The test system consists of 150mL of test culture medium + 30mL of activated bacterial agent after centrifugation.

[0037] The method for counting miscellaneous bacteria (only heterotrophic bacteria are counted) is the dilution plate counting method.

[0038] Table 1. Results of co-fermentation of Candida albicans and nitrite-oxidizing bacteria in a 5L tank.

[0039]

[0040]

[0041] Example 2: Effect of Low-Temperature Treatment of Seed Liquid on Fermentation Efficiency

[0042] Referring to Example 1, this example further illustrates the positive effect of low-temperature treatment on high-density fermentation of nitrite oxidation enrichment broth through the following process: The specific experimental process is as follows:

[0043] (1) Prepare NOB fermentation medium with a volume of 3L / 5L. NOB fermentation medium: NaCl 0.3g / L, MgSO4·7H2O 0.2g / L, K2HPO4 0.1g / L, organic matter 1g / L, trace element stock solution 1mL / L, carrier 1g / L. Sterilize at 121℃ for 30min and adjust pH to 7.0.

[0044] (2) Introduce the nitrite oxidation enrichment solution into the sterilized NOB fermentation medium, add sodium nitrite to make the final concentration of nitrite nitrogen reach 100 mg / L. Whenever the nitrite nitrogen in the system is consumed, add sodium nitrite to make the final concentration of nitrite nitrogen reach 100 mg / L. After 5 batch feedings, the culture is ended. The seed liquid sampling rate is 110 ± 15 mg NO2-N / L / h.

[0045] (3) After the six groups of seed cultures were refrigerated at 4℃ for different durations (see Table 2), they were inoculated at 10% of the seed culture and cultured in sterilized NOB fermentation medium. 48 hours before the previous addition of nitrite nitrogen was consumed, a certain amount of sodium nitrite was immediately added to bring the final concentration of nitrite nitrogen in the tank to 100 mg / L. 48 hours after the previous addition of nitrite nitrogen was consumed, a certain amount of sodium nitrite was immediately added to bring the final concentration of nitrite nitrogen in the tank to 300 mg / L. The fermentation conditions and feed amounts were consistent for all groups until the fermentation rate stopped increasing, at which point fermentation was terminated.

[0046] Table 2. Fermentation rates of seed culture after inoculation under different low-temperature treatment conditions.

[0047]

[0048] Note: The rate units in the table are all mg NO2-N / L / h; in addition, since fermentation generally requires several stages of scale-up and becomes less stable with more passages, this example uses two passages in shake flasks before starting fermentation to simulate the step-by-step transfer process in actual scale-up.

[0049] Table 2 shows the nitrite oxidation rates after 48 hours of fermentation and after stopping the fermentation under different refrigeration conditions. The experimental results show that the low-temperature treatment before inoculation has a positive effect on the activity of nitrite-oxidizing bacteria in the enrichment solution, and this operation can increase the efficiency of fermentation in the middle and late stages.

[0050] Example 3: Inhibitory effect of different concentrations of nitrite on Candida albicans

[0051] Given that nitrite is toxic to many microorganisms, and in the high-density fermentation of this invention, Candida albicans needs to have a certain nitrite tolerance in order to compete for nutrients with known contaminating bacteria, and also needs to be sensitive to high concentrations of nitrite in order to remove it in the later stages of fermentation. In this example, Escherichia coli was used as a control, and the inhibitory effect of different concentrations of nitrite on Candida albicans was further verified through the following process.

[0052] Prepare LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7.2-7.5), sterilize at 121℃ for 20 min, and then culture Escherichia coli.

[0053] Prepare YPD medium (1% yeast extract, 2% peptone, 2% glucose, natural pH), sterilize at 115℃ for 15 min, and then culture Candida albicans.

[0054] Add nitrite nitrogen (specifically sodium nitrite) according to the concentrations in Table 3, and inoculate Escherichia coli or Candida seed culture in the logarithmic growth phase at 1%. After incubation in a shaker until the stationary phase, spread the bacterial count and calculate the bacterial count loss rate after adding sodium nitrite.

[0055] Table 3. Detection results of nitrite tolerance in Escherichia coli and Candida albicans.

[0056] As shown in Table 3, nitrite levels below 300 mg N / L have no significant inhibitory effect on Escherichia coli, while nitrite levels between 400 and 500 mg N / L have a partial inhibitory effect on Escherichia coli. Nitrite levels below 100 mg N / L have no significant inhibitory effect on Candida albicans, but more than half of Candida albicans can survive at 200 mg / L nitrite levels, and Candida albicans cannot survive at 300 mg / L nitrite levels.

[0057] Example 4: Effects of different Candida inoculation amounts on fermentation efficiency

[0058] This example compares the effect of Candida albicans inoculation amount on the fermentation effect of nitrite oxidation enrichment broth. The specific experimental procedure is as follows:

[0059] (1) Preparation of NOB fermentation medium: NaCl 0.3g / L, MgSO4·7H2O 0.2g / L, K2HPO4 0.1g / L, organic matter 1g / L, trace element mother liquor 1mL / L, carrier 1g / L, sterilized at 121℃ for 30min and then adjusted pH to 7.0.

[0060] (2) Preparation and low-temperature treatment of seed culture: The nitrite oxidation enrichment solution was inoculated into sterilized NOB fermentation medium, and sodium nitrite was added to bring the final concentration of nitrite nitrogen to 100 mg / L. Whenever the nitrite nitrogen in the system was consumed, sodium nitrite was added again to bring the final concentration of nitrite nitrogen to 100 mg / L. The culture was terminated after 5 batch feedings. The seed culture collection rate was 110 ± 15 mg NO2-N / L / h. The seed culture was stored at 4℃ for 7 days.

[0061] (3) Fermentation: After inoculating the seed culture at 10%, the *Candida* culture (obtained by culturing *Candida* in YPD medium to the logarithmic growth phase) was centrifuged and resuspended in fermentation medium. This resuspension was repeated twice before inoculation into the fermentation medium at inoculation rates of 0.1% and 0.5% (with no inoculation as a control). After inoculation, the culture was placed in a shaker at 30°C and cultured with feed as usual, following the same process as in Example 1.

[0062] The test results are shown in Table 4. As can be seen from Table 4, after adding Candida albicans, the fermentation rate of nitrite oxidizing bacteria increased and the proportion of miscellaneous bacteria decreased to a certain extent. Moreover, the nitrite oxidation rate, the change in the amount of miscellaneous bacteria and the inoculation amount of Candida albicans were all correlated.

[0063] Table 4. Results of co-culture of Candida albicans and nitrite-oxidizing bacteria in shake flasks.

[0064]

[0065] Comparative Example 1: Effect of Organic Matter in Fermentation Medium on Fermentation Efficiency

[0066] To further verify the necessity of co-culturing nitrite-oxidizing bacteria with Candida albicans in nitrite-oxidizing bacteria fermentation, this example designed a control experiment in which nitrite-oxidizing bacteria were cultured alone with the culture medium having organic matter removed. The procedure is as follows:

[0067] (1) Preparation of NOB fermentation medium without organic matter: NaCl 0.3g / L, MgSO4·7H2O 0.2g / L, K2HPO4 0.1g / L, organic matter 1g / L, trace element stock solution 1mL / L, carrier 1g / L, sterilize at 121℃ for 30min and adjust pH to 7.0, the liquid volume is 3L / 5L.

[0068] (2) Preparation and low-temperature treatment of seed culture: The nitrite oxidation enrichment solution was inoculated into sterilized NOB fermentation medium, and sodium nitrite was added to bring the final concentration of nitrite nitrogen to 100 mg / L. Whenever the nitrite nitrogen in the system was consumed, sodium nitrite was added again to bring the final concentration of nitrite nitrogen to 100 mg / L. The culture was terminated after 5 batch feedings. The seed culture collection rate was 110 ± 15 mg NO2-N / L / h. The seed culture was stored at 4℃ for 7 days.

[0069] (3) Fermentation: After inoculating the seed culture at 10%, fermentation was started in sterilized NOB fermentation medium (using the NOB fermentation medium from Example 1 as the control group). For the first 48 hours, a certain amount of sodium nitrite was added immediately after the previously added nitrite was consumed, bringing the final nitrite concentration in the tank to 100 mg / L. After 48 hours, a certain amount of sodium nitrite was added immediately after the previously added nitrite was consumed, bringing the final nitrite concentration in the tank to 300 mg / L. Fermentation conditions and feed amounts were consistent across all groups. Fermentation was stopped when the rate no longer increased.

[0070] The test results are shown in Table 5. Table 5 shows that removing organic matter from the culture medium does not weaken the reproduction of denitrifying bacteria in the later stages; on the contrary, it worsens the fermentation results of nitrite oxidizing and reducing bacteria. The reason for this is speculated to be that, under high nitrate concentrations, some denitrifying bacteria will use the metabolic products and cells of nitrite-oxidizing bacteria as organic matter for growth.

[0071] Therefore, it is meaningful to add Candida albicans to help metabolize organic matter in the early stages of nitrite-oxidizing bacteria cultivation.

[0072] Table 5. Results of the experiment on the effect of organic matter in the culture medium on fermentation.

[0073]

[0074] Example 5: Degradation capacity test of nitrite-oxidizing bacterial inoculant

[0075] This example uses fishpond aquaculture wastewater as the test subject to test the degradation ability of the nitrite-oxidizing bacterial agent prepared by the method of this invention. The operation process in this example is as follows:

[0076] Inoculant 1 was prepared from the control group without low-temperature treatment in Example 1 (i.e., seeds were not treated at low temperatures and were not co-cultured with Candida albicans), and inoculant 2 was prepared from the experimental group in Example 1 (i.e., low-temperature treatment + 0.5% inoculation with Candida albicans).

[0077] After activating the two bacterial agents for 3 hours, they were added to the fishpond aquaculture wastewater. The specific experimental conditions were: dissolved oxygen was kept above 5 mg / L, temperature was 30℃, the initial nitrite nitrogen concentration in the wastewater was 2 mg / L, and the bacterial agent dosage was 1‰.

[0078] The control group (CK) was used as the control group without any added microbial agents. There were three groups in total, each of which included three parallel groups. The degradation rate was recorded at 6h, 24h, 31h and 48h, respectively. The test results are shown in Table 6.

[0079] Table 6. Degradation results of nitrite in fishpond aquaculture wastewater by different microbial agents.

[0080]

[0081] Note: The unit for nitrite concentration in the table is mg / L.

[0082] As shown in Example 1, the rate of biochemical agent 1 was 140.98 mg NO2-N / L / h, and the rate of biochemical agent 2 was 279.78 mg NO2-N / L / h. Table 6 shows that although both biochemical agents 1 and 2 could reduce nitrite in wastewater to below 0.1 mg / L, the biochemical agent fermented from seeds treated at low temperature and combined with Candida albicans degraded nitrite in actual wastewater at a faster rate, completely degrading 2 mg / L of nitrite within 31 hours.

[0083] In summary, this invention effectively improves the fermentation level of nitrite-oxidizing bacteria enrichment solution by coordinating the pretreatment of seed liquid before fermentation, the introduction of Candida albicans in the early stage of fermentation, and the feeding of high-concentration nitrite nitrogen in the middle and late stages of fermentation. It also obtains a bacterial agent product with a significantly improved fermentation rate, solving the technical problem of low culture rate of nitrite-oxidizing bacteria that cannot be cultured in pure form in the prior art.

[0084] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A high-density fermentation method for a nitrite-oxidizing bacterial enrichment solution, characterized in that, Includes the following steps: S1. Prepare nitrite-oxidizing bacteria seed solution from nitrite-oxidizing bacteria enrichment solution and refrigerate the nitrite-oxidizing bacteria seed solution. S2. The refrigerated nitrite-oxidizing bacteria seed culture was inoculated into a sterilized fermentation medium, and yeast was simultaneously inoculated for fermentation. The fermentation process consisted of two stages: In the early stage of fermentation, whenever the nitrite nitrogen in the fermentation system is consumed, nitrite nitrogen is added and its final concentration in the fermentation medium is ≤100mg / L. During the middle and late stages of fermentation, whenever the nitrite nitrogen in the fermentation system is consumed, nitrite nitrogen is added and its final concentration in the fermentation medium is ≥300 mg / L. Fermentation is terminated when the fermentation rate no longer increases. The nitrite-oxidizing bacterial enrichment solution has Nitrobacterium velutipes as the dominant bacterial species, and Nitrobacterium velutipes accounts for more than 70% of the total bacterial count in the enrichment solution. The yeast is Candida albicans; The refrigeration process described in step S1 takes 3-15 days.

2. The high-density fermentation method for nitrite-oxidizing bacterial enrichment broth according to claim 1, characterized in that, In step S2, the inoculation amount of the nitrite-oxidizing bacteria seed solution is 10-15%, and the inoculation amount of yeast is 0.1-0.5%.

3. The high-density fermentation method for nitrite-oxidizing bacterial enrichment broth according to claim 1, characterized in that, The fermentation medium contains organic matter, and the organic matter is yeast extract, glucose and / or glycerol.

4. The high-density fermentation method for nitrite-oxidizing bacterial enrichment broth according to claim 3, characterized in that, The fermentation medium consists of: NaCl 0.3 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 0.1 g / L, organic matter 1 g / L, trace element mother liquor 1 mL / L, and carrier 1 g / L.

5. The microbial agent product prepared by the high-density fermentation method of the nitrite-oxidizing bacteria enrichment solution according to any one of claims 1 to 4.

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