Strain with high nitrite tolerance and ammonium production function, microbial inoculum and preparation method and application of microbial inoculum
By developing a microbial agent from the highly nitrite-tolerant strain Streptomyces anulatus 255, the harmful effects of high concentrations of nitrite on microorganisms and the rhizosphere microecology of plants have been solved, achieving efficient nitrogen conversion and resource utilization, and breaking through the bottleneck of traditional denitrification processes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG UNIV
- Filing Date
- 2025-10-13
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, high concentrations of nitrite are harmful to microorganisms and the balance of plant rhizosphere microecology, and traditional denitrification processes have poor tolerance, leading to nitrogen resource waste and environmental pollution.
A strain of Streptomyces anulatus 255 with high nitrite tolerance and ammonium production function was developed. It was prepared as a bacterial agent by mixing with trehalose, glycerol, D-ascorbic acid, potassium sorbate and sodium dehydroacetate for the treatment of nitrite-containing wastewater and ecological restoration.
It achieves efficient conversion of nitrates and nitrites into ammonium nitrogen, improves nitrogen utilization, reduces operating costs, and is suitable for aquaculture, industrial wastewater treatment, and soil remediation.
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Figure CN122012299A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a bacterial strain and a bacterial agent, as well as a method for preparing the bacterial agent and its application, belonging to the field of microbial technology. Background Technology
[0002] Ammonium nitrogen is one of the main nitrogen sources in soil. For microorganisms in the environment, ammonium nitrogen is a direct and scarce nitrogen source, especially for those microorganisms that cannot directly use nitrate as a nitrogen source. Ammonium nitrogen is also an important nitrogen source for plants. When plants use ammonium nitrogen as a nitrogen source, they release hydrogen ions to improve the alkaline environment of the soil. Furthermore, plants utilize ammonium ions more efficiently than nitrates, which is a significant advantage of ammonium nitrogen. With the continuous expansion of industrial and agricultural wastewater, livestock and poultry farming tailwater, and urban sewage treatment systems, nitrite has become one of the main causes of eutrophication and public health risks. Nitrite can inhibit the oxygen-carrying function of hemoglobin, and its acute lethal concentration in aquaculture is only [missing information]. High concentrations of nitrite not only disrupt the rhizosphere microecological balance but also cause soil nitrogen loss through denitrification. Emissions. Therefore, it is necessary to develop technologies that can stably survive under high nitrite stress and efficiently convert nitrogen into ammonium (ammonium) that can be absorbed and utilized by crops. Functional strains are particularly important.
[0003] At the application level, bio-agents have been proven to have the following advantages: ① In aquaculture waters, they can rapidly convert acutely toxic nitrites into ammonium that can be utilized by phytoplankton, reducing water exchange frequency and wastewater discharge; ② In soil remediation, inoculating functional microbial agents using biofortification technology can maintain rhizosphere nitrogen cycle homeostasis under high nitrite stress, improve crop nitrogen use efficiency, and reduce nitrogen loss. Discharge; ③ Compared with conventional nitrification-denitrification processes, microbial agent processes do not require strict staged control, have low operating costs and low technical requirements, and are suitable for decentralized rural sewage treatment and in-situ purification of aquatic wastewater from facilities. Summary of the Invention
[0004] To address the problems of low nitrogen availability, excessive use of chemical nitrogen fertilizers, soil ecological degradation, and poor tolerance to traditional denitrification processes, this invention proposes a strain and agent with high nitrite tolerance and ammonium production function, as well as a method for preparing and applying the agent.
[0005] The technical solution adopted by the present invention to solve the above problems is as follows: A strain with high nitrite tolerance and ammonium production function is named *Streptomyces circumvallate*. Streptomyces anulatus Accession number 255 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 12, 2025, with accession number CCTCCNO: M 20251026.
[0006] The present invention discloses a microbial agent with high nitrite tolerance and ammonium production function, which is prepared by concentrating the fermentation broth of Streptomyces anulatus 255 and mixing it with trehalose, glycerol, D-ascorbic acid, potassium sorbate and sodium dehydroacetate. Specifically, after concentrating each liter of fermentation broth to 150 mL, 4 g of trehalose, 2 g of glycerol, 0.5 g of D-ascorbic acid, 0.3 g of potassium sorbate and 0.04 g of sodium dehydroacetate are added, and the volume is adjusted to 200 mL.
[0007] The preparation method of the microbial agent with high nitrite tolerance and ammonium-producing function described in this invention includes the following steps:
[0008] Step 1: Inoculate Streptomyces anulatus 255 into LB broth medium and culture.
[0009] Step 2: Resuspend and add a protectant to prepare a bacterial agent.
[0010] Furthermore, in step 1, the temperature of the LB liquid medium was 30°C, the incubation time was 48 hours, and the viable bacteria concentration in the inoculum was 2.5 × 10⁻⁶. 8 cfu / mL.
[0011] Furthermore, in step 1, the temperature of the LB liquid medium was 30°C, the incubation time was 48 hours, and the viable bacteria concentration in the inoculum was 2.5 × 10⁻⁶. 8 cfu / mL.
[0012] The present invention relates to the application of a bacterial agent with high nitrite tolerance and ammonium-producing function, which is used for the treatment of nitrite-containing wastewater, nitrogen recovery, and ecological restoration.
[0013] The beneficial effects of this invention are:
[0014] 1. The strain described in this invention can be used in scenarios such as aquaculture tailwater, industrial high-salt wastewater, and eutrophic water bodies to achieve efficient conversion of nitrate and nitrite and resource utilization of ammonium nitrogen. It is applicable to the fields of environmental microbiology technology, biological denitrification, agricultural circular economy and ecological restoration technology.
[0015] 2. This invention uses plant rhizosphere soil and polluted water as sources to isolate a strain with ammonium production and nitrite tolerance. Its ammonium production and nitrite tolerance are determined through culture medium experiments and simulated soil tests. Finally, it is made into a bacterial agent to overcome the bottleneck of poor tolerance and nitrogen waste in traditional denitrification processes, and provides a green and sustainable innovative path for nitrogen pollution control and nitrogen resource recycling in water bodies. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the ammonium production pathway in Streptomyces anulatus 255;
[0017] Figure 2 This is a schematic diagram of the colony morphology of Streptomyces anulatus 255.
[0018] Figure 3 This is a schematic diagram of the phylogenetic tree of Streptomyces anulatus 255, where XX3 is Streptomyces anulatus 255 of this invention;
[0019] Figure 4 This is a schematic diagram illustrating the activity test of Streptomyces anulatus 255 in nitrite medium.
[0020] Figure 5 This is a schematic diagram of the ammonium production curve of Streptomyces anulatus 255 in nitrate medium;
[0021] Figure 6 This is a schematic diagram of the ammonium production curve of Streptomyces anulatus 255 in anaerobic nitrate medium;
[0022] Figure 7 This is a schematic diagram of the ammonium production curve of Streptomyces anulatus 255 in nitrite medium;
[0023] Figure 8 This is a schematic diagram showing the changes in soil nitrogen after applying Streptomyces anulatus 255 inoculant.
[0024] Figure 9 This is a schematic diagram illustrating the impact on plant physiological and biochemical data.
[0025] Figure 10 This is a schematic diagram of the nitrate nitrogen standard curve obtained by spectrophotometry using thymol;
[0026] Figure 11 This is a schematic diagram of the nitrite standard curve obtained by sulfonamide spectrophotometry.
[0027] Figure 12 This is a schematic diagram of the ammonium nitrogen standard curve obtained by the salicylic acid-sodium dichloroisocyanurate method. Detailed Implementation
[0028] Example 1
[0029] (1) Isolation and screening of strains
[0030] After obtaining soil samples using a sampler, weigh the soil samples and perform serial dilutions with 9 times their volume of water. Shake the samples overnight at 30°C and 150 rpm / min. After serial dilution, take 200 μL of the sample and spread it on a denitrification plate containing nitrite. Incubate the plate at 30°C for 3-5 days.
[0031] Single colonies were picked from the petri dishes and incubated on LB broth for 48 hours. The colonies were then streaked three times on denitrification plates until a uniform colony morphology was observed. The single colonies were then transferred to LB broth for enrichment, washed with sterile water, and then frozen in 30% glycerol at -80°C.
[0032] (2) Functional verification
[0033] The glycerol bacteria obtained in the previous step were re-inoculated into LB medium for activation. After washing and resuspending with sterile water, they were inoculated again into nitrate liquid medium and nitrite liquid medium at an inoculation rate of 2%. Three replicates were set up for each group and placed in shaker and anaerobic fermenter for culture. After 72 hours, samples were taken to determine the ammonium nitrogen content in the medium.
[0034] LB medium formulation: 5g yeast extract, 10g tryptic peptone, 10g sodium chloride, adjust pH to 7.0 with 2mol / L NaOH.
[0035] Nitrate medium formula: NaNO3 0.85g / L, KH2PO4 1.5g / L, K2HPO4 4g / L, MgSO4 0.02g / L, monohydrate and glucose 6.93g / L, autoclaved at 115℃ for 30min.
[0036] Nitrite culture medium formula: NaNO2 0.69g / L, KH2PO4 1.5g / L, K2HPO4 4g / L, MgSO4 0.02g / L, monohydrate and glucose 6.93g / L, autoclaved at 115℃ for 30min.
[0037] (3) Strain identification
[0038] After activating the screened bacteria, genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit. PCR amplification of 16S rDNA was performed using each bacterial genome as a template. The primer sequences were 27F: TAGGYTACCTTGTTACGACT and 1492R: AGAGTTTGATCMTGGCTCAG. The PCR reaction system is shown in Table 1, and the PCR amplification conditions are shown in Table 2. After confirming the amplification products were correct by 1.0% agarose gel electrophoresis, they were recovered using an agarose gel DNA recovery kit and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were submitted to the GenBank database. The 16S rDNA sequences of the screened bacteria were aligned to the NCBI nucleic acid sequence database using BLASTN. A phylogenetic tree (e.g., phylogenetic tree) was constructed using the Neighbor-Joining Algorithm (NJ) method with MEGA11 software. Figure 3 (As shown). Comparison shows that several strains with high homology with it are all Streptomyces, therefore this strain can be identified as Streptomyces.
[0039]
[0040] Example 2
[0041] The Streptomyces strain from Example 1 was subjected to a nitrite tolerance test to determine its nitrite tolerance ability.
[0042] The strain was inoculated at a 2% inoculum in nitrite medium and cultured for 24 hours. Then, 5 μL of sodium resazurin solution was added to the nitrite liquid medium, and the color of the medium was observed after 2 hours.
[0043] The activity of the strain was determined by inoculating it into nitrite liquid medium and observing a color change from blue to red. Figure 4 As shown.
[0044] Example 3
[0045] Ammonium production capacity test of Streptomyces from Example 1
[0046] After activation with LB liquid medium, the culture was washed with an equal volume of sterile water. 200 μL of the bacterial culture was then inoculated into 4 ml of nitrate and nitrite medium, respectively. An anaerobic group was also set up in the nitrate medium. Each group was incubated in triplicate. The culture was incubated at 30°C for 48 hours, and the culture was zeroed using uninoculated medium. The ammonium nitrogen content in the medium was determined every 12 hours using the sodium diisochlorocyanurate method, and an ammonium production curve was plotted. Figure 5-7 As shown.
[0047] Example 4
[0048] Take 10 μL of glycerol bacteria and inoculate it into LB liquid medium. Incubate at 30°C and 180 rpm / min for 48 hours.
[0049] Inoculate the bacterial culture into the fermenter at a 5% inoculum rate. The fermenter should be filled with 50% liquid. Incubate at 30°C for 2-3 days.
[0050] The fermentation broth formula is: 20g / L soluble starch and 15g / L soybean meal. Add 3g / L NaCl and 2g / L NaCl. Adjust the pH to 7.0-7.2 using 2mol / L NaOH, and then add water to a final volume of 1L.
[0051] Wash 1L of fermentation broth with distilled water and concentrate it to 150mL. Add 4g of trehalose, 2g of glycerol, 0.5g of D-ascorbic acid, 0.3g of potassium sorbate, and 0.04g of sodium dehydroacetate, and make up to 200mL to prepare the inoculum.
[0052] Example 5
[0053] Tests were conducted on changes in soil nitrogen levels after inoculation with Streptomyces circinus.
[0054] Streptomyces was prepared into an inoculum and applied to soil supplemented with 50% urea. A 5 cm layer of vermiculite was then laid at the bottom of the soil, followed by a 5 cm layer of soil with half the inoculum added, and then another 5 cm layer of soil with the remaining inoculum added. A control group without inoculum application was used, with three replicates. Two soil types were selected: CK1 (saline-alkali soil) and CK2 (black soil from cornfields). Every fifteen days, whole soil samples were taken using a sampler, diluted with nine times their weight of water, and shaken on a shaker at 30°C and 150 rpm for 1 hour. 500 μL of the shaken solution was centrifuged at 12000 rpm for 2 minutes. The supernatant was used to determine nitrate nitrogen, ammonium nitrogen, and nitrite nitrogen using the N-ethylenediamine spectrophotometric method, the sodium salicylate-diisochlorocyanurate method, and the sulfonamide spectrophotometric method, respectively. Specific information is as follows: Figure 8 As shown.
[0055] Nitrate Detection - Thymol Spectrophotometry
[0056] Ammonium aminosulfonate solution: Weigh 2g of ammonium aminosulfonate and dilute to 100mL with 1+4 acetic acid.
[0057] Thymol solution: Add 0.5g of thymol to anhydrous ethanol and bring the volume to 100mL.
[0058] Silver sulfate solution: 1g of silver sulfate is diluted to 100mL with concentrated sulfuric acid.
[0059] Add 200 μL of detection solution to 40 μL of ammonium aminosulfonate solution, mix well and let stand for 5 min. Add 80 μL of thymol solution and 800 μL of silver sulfate solution, let stand for 5 min, add 3.2 mL of water to dilute, slowly add 3.2 mL of concentrated ammonia solution, add water to make up to 10 mL, and after cooling, perform photometric measurement at 415 nm.
[0060] Weigh 2g of sodium nitrate and dilute to 100mL. Add 1μL, 0.75μL, 0.5μL, and 0.25μL of water and test solution to make up the volume. Plot a standard curve using the obtained data as shown below. Figure 10 As shown.
[0061] Nitrite Detection - Sulfonamide Spectrophotometric Method
[0062] Sulfonamide solution: Add 4g sulfonamide to 30mL water and 10mL phosphoric acid to make up to 50mL.
[0063] N-(1-naphthyl)-ethylenediamine solution: Add 0.4g N-(1-naphthyl)-ethylenediamine to water and bring the volume to 100mL.
[0064] Colorimetric solution: 20 mL sulfonamide solution plus 20 mL N-(1-naphthyl)-ethylenediamine solution plus 20 mL phosphoric acid.
[0065] Add 4 mL of water and μL of colorimetric reagent to 160 μL of sample and let stand for 20 min. Then, perform photometric measurements at 540 nm.
[0066] Weigh 0.1g of sodium nitrite and dilute to 100mL. Pipette 2μL, 1.5μL, 1μL, and 0.5μL of the solution to make up the difference between water and the test solution. Plot the obtained data to create a standard curve as shown below. Figure 11 As shown.
[0067] Ammonium nitrogen detection - sodium salicylate and sodium dichloroisocyanurate method
[0068] Sodium nitrosoferricyanide salicylate solution: Weigh 0.64g NaOH, 12.8g sodium salicylate, and 0.14g sodium nitrosoferricyanide, and add water to make up to 80mL.
[0069] Sodium dichloroisocyanurate solution: Add 0.09g of sodium dichloroisocyanurate to water and bring the volume to 80mL.
[0070] Add 80 μL of sodium salicylate nitrosoferricyanide solution and 20 μL of sodium dichloroisocyanurate solution to 400 μL of sample, let stand for 30 min, and then perform photometric measurement at 660 nm.
[0071] Weigh 0.1g of ammonium chloride and dilute to 100mL. Pipette 1μL, 0.75μL, 0.5μL, and 0.25μL of the solution to make up the difference between water and the test solution. Plot the obtained data to create a standard curve as shown below. Figure 12 As shown.
[0072] Example 6
[0073] In addition to the methods used in Example 5, rapeseed seeds of variety Zhongshuang 9 (Brassica napus), numbered GPD (2017) 420055, were also planted. Two types of soil were selected, with three controls and three treatments for each soil type. Five seeds were sown in each treatment. On days 15 and 30, one plant was selected from each sample to measure plant height, leaf nitrogen content, and dry weight. Figure 9 As shown.
[0074] Plant height measurement: Use a ruler to measure the vertical distance from the soil surface to the highest point of the main stem when it is naturally upright.
[0075] Leaf nitrogen content: Select the top four leaves, measure the SPAD value using a SPAD meter, and then convert it to nitrogen content using a formula.
[0076] SPAD×0.039 - 0.47 ≈ Leaf nitrogen (%, DW)
[0077] Dry weight measurement: After soaking the soil with water, loosen the soil continuously, remove the plant with its entire root system from the soil at once, rinse until no soil is left, dry at 105℃ to constant weight, and measure the dry weight using an analytical balance.
[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.
Claims
1. A strain with high nitrite tolerance and ammonium production function, characterized in that, The strain was named *Streptomyces circumvallate*. Streptomyces anulatus 255, deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M 20251026.
2. The strain with high nitrite tolerance and ammonium production function according to claim 1, characterized in that, The ammonium production function is manifested in the continuous and stable production of ammonium nitrogen under the conditions of nitrate or nitrite as nitrogen source. Its average ammonium production efficiency is 3.1-4.3 mg / (L·h) under nitrate conditions and 0.92-1.02 mg / (L·h) under nitrite conditions.
3. A bacterial agent prepared using the strain described in claim 1, characterized in that, Streptomyces circumvallate Streptomyces anulatus After the fermentation broth of 255 was concentrated, it was mixed with trehalose, glycerol, D-ascorbic acid, potassium sorbate and sodium dehydroacetate. Specifically, after each liter of fermentation broth was concentrated to 150 mL, 4 g of trehalose, 2 g of glycerol, 0.5 g of D-ascorbic acid, 0.3 g of potassium sorbate and 0.04 g of sodium dehydroacetate were added, and the volume was adjusted to 200 mL.
4. A method for preparing a bacterial agent with high nitrite tolerance and ammonium-producing function, characterized in that, The specific steps include: Step 1: Sterilomyces circumvallate Streptomyces anulatus 255 was inoculated into LB bacterial culture medium and cultured. Step 2: Resuspend and add a protectant to prepare a bacterial agent.
5. The preparation method of a microbial agent with high nitrite tolerance and ammonium-producing function according to claim 3, characterized in that, In step 1, the temperature of the LB liquid medium was 30℃, the incubation time was 48 hours, and the viable bacteria concentration in the inoculum was 2.5 × 10⁻⁶. 8 cfu / mL.
6. The preparation method of a microbial agent with high nitrite tolerance and ammonium-producing function according to claim 3, characterized in that, The protective agents in step 2 are trehalose, glycerol, D-ascorbic acid, potassium sorbate, and sodium dehydroacetate.
7. The application of a bacterial agent with high nitrite tolerance and ammonium-producing function, characterized in that, The bacterial agent is used for the treatment of nitrite-containing wastewater, nitrogen recovery, and ecological restoration.
8. The application of a bacterial agent with high nitrite tolerance and ammonium-producing function, characterized in that, When applied to the soil, the soil ammonium nitrogen content increased by 2.19-3.05 times in 15 days and by 1.44-1.64 times in 30 days. The plant dry weight increased by 12-35%, the plant height increased by 10-30%, and the leaf nitrogen content increased by 38-43%.