Strain for removing ammonia nitrogen and application thereof

Abstract

The invention discloses ginseng tukast ranilla and application, the ginseng tukast ranilla has the name of IURM-H19, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has the preservation number of CGMCC No.29727, and has the preservation date of January 23, 2024; according to the application disclosed by the invention, ammonia nitrogen in sewage can be degraded, and COD (Chemical Oxygen Demand) in the sewage also can be well degraded; according to the present invention, with the application of the ginseng tukast ranilla IURM-H19, the corresponding biological agent can be developed, and can be used for degrading ammonia nitrogen and COD in environmental sewage, preventing water eutrophication, and reducing the harmful effect on human and aquatic organisms.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation, and specifically relates to a strain for removing ammonia nitrogen and application thereof. Background Art

[0002] Whether in a high or low concentration state, when the ammonia nitrogen content in the water system is too high, it will bring different degrees of harm, mainly as follows: (1) Ammonia nitrogen will consume a large amount of oxygen dissolved in the water environment, causing the water to deteriorate. In severe cases, it may even turn black and smelly, and the water quality will deteriorate, posing a threat to the organisms living in it; (2) Ammonia nitrogen can react with substances in the water system to generate secondary pollutants, causing secondary pollution, and its treatment will increase the treatment cost; (3) Too much nitrogen content in the water will lead to eutrophication of the water body, causing an increase in the number of algae and accelerating the rate of water deterioration; (4) Nitrite nitrogen and nitrate nitrogen substances in the water will cause greater harm to humans and aquatic organisms. Some of these substances can cause cancer, mutations, and teratogenesis when accumulated in large quantities. In addition, when the ammonia nitrogen content in the water is too high, it will also cause major incidents such as poisoning of humans and animals and "involvement" of related waters. Based on these factors, the treatment of ammonia nitrogen in the water body is not only a hot topic, but also a necessary part of environmental protection.

[0003] In recent years, nitrogen compounds in the environment have continued to accumulate, especially in water pollution. When nitrogen compounds enter the water and are converted into more stable secondary pollutants through chemical changes, they will have a greater impact on water resources. As the water resource crisis increasingly threatens the health of organisms and humans, people's environmental awareness is also increasing, and they are gradually aware of the importance of treating nitrogen-containing wastewater, especially treating ammonia nitrogen-containing wastewater.

[0004] Using microbial methods to degrade nitrogen-containing substances in wastewater and exploring the metabolic laws of strains can provide a certain reference value for the engineering application of strains, which is of great significance for protecting the water environment and alleviating the water resource crisis.

[0005] At present, the commonly used methods for removing ammonia nitrogen are mainly: physical precipitation, chemical precipitation, and biological methods. Compared with mainstream methods such as physical and chemical methods, biological degradation of ammonia nitrogen has significant advantages, mainly reflected in high cost-effectiveness, environmental friendliness, stable treatment effect, strong resource recovery ability, strong treatment capacity, high sustainability, good flexibility, strong adaptability, mature technology and a wide range of treatment. New denitrification technologies such as short-range nitrification and denitrification and anaerobic ammonia oxidation have strict requirements on process conditions in practical applications, especially the requirements for dissolved oxygen are more stringent. Therefore, how to obtain a strain that has good effect in treating ammonia nitrogen in wastewater and short degradation cycle has become a problem to be solved. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a ginseng soil castellaniella.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Castellaniella ginsengisoli IURM H19 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 23, 2024, with a deposit number of CGMCC No.29727, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a microbial agent containing Castellaniella ginseng.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of Castellania ginsengensis or a bacterial agent containing Castellania ginsengensis in removing ammonia nitrogen from waste liquid.

[0012] As a preferred embodiment of the application of the present invention, Castellania ginsengensis or a bacterial agent containing Castellania ginsengensis is added to wastewater containing ammonia nitrogen to carry out a reaction.

[0013] As a preferred embodiment of the application of the present invention, the OD600 value of the Castellania ginseng added to the wastewater is at least 0.59.

[0014] As a preferred embodiment of the application of the present invention, the reaction is carried out at 25-40°C, 180-220 rpm, and pH 6-8 for 0.5h-72h.

[0015] As a preferred embodiment of the application of the present invention, the reaction is carried out at 30-35°C, 180rpm and pH 7 for 36 hours.

[0016] As a preferred embodiment of the application of the present invention, the ammonia nitrogen concentration in the wastewater is 300-500 mg / L.

[0017] As a preferred embodiment of the application of the present invention, the waste water is waste water from the production process of erythromycin.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of Castellaniella ginseng in the preparation of a product for removing ammonia nitrogen from wastewater.

[0019] Beneficial effects of the present invention:

[0020] 1. The present invention discovers for the first time a ginseng soil Castellaniella, which uses a biological treatment method to degrade ammonia nitrogen and has the advantages of high deodorization, high biodegradation efficiency, no secondary pollution and low cost.

[0021] 2. The present invention improves the efficiency of ammonia nitrogen degradation of the strain by optimizing the culture conditions.

[0022] 3. The Castellania ginseng IURM-H19 of the present invention can be used to develop corresponding biological preparations for the degradation of ammonia nitrogen and COD in environmental sewage, preventing eutrophication of water bodies and reducing their harmful effects on humans and aquatic organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 This is a crystal violet single staining image of Castellaniella ginsengisoli IURM H19 in Example 2 of the present invention.

[0025] Figure 2 This is a colony morphology diagram of Castellaniella ginsengisoli IURM H19 in Example 2 of the present invention.

[0026] Figure 3 This is a diagram of the PCR electrophoresis results in Example 3 of the present invention.

[0027] Figure 4 This is the phylogenetic tree diagram established in Example 3 of the present invention.

[0028] Figure 5 This is the ammonia nitrogen standard curve diagram in Example 4 of the present invention.

[0029] Figure 6This is a dynamic curve diagram of ammonia nitrogen degradation by IURM H19 in Example 4 of the present invention.

[0030] Figure 7 This is a diagram showing the optimization of pH conditions for ammonia nitrogen degradation by IURM H19 in Example 5 of the present invention.

[0031] Figure 8 This is a diagram showing the optimization of the temperature conditions for ammonia nitrogen degradation by IURM H19 in Example 6 of the present invention.

[0032] Fig. 9 The removal efficiency of IURM H19 on COD in pig manure wastewater in Example 7 of the present invention is DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0036] The raw materials and reagents used in the present invention are shown in Table 1.

[0037] Table 1

[0038]

[0039] Crystal violet staining:

[0040] First, use a pipette to absorb an appropriate amount of bacterial solution onto a glass slide, gently spread the bacterial solution, and dry it with a low flame. Secondly, when a layer of bacterial film is formed on the glass slide, add 1 to 2 drops of crystal violet for staining. After staining for 1 to 2 minutes, rinse off the dye with a slow stream of water, and then use absorbent paper to absorb the residual moisture around. Finally, perform a microscopic examination. After finding the field of view under a low-power microscope, use a high-power microscope to observe the specific morphological characteristics of the strain.

[0041] Unless otherwise stated, all experimental methods disclosed in the present invention adopt conventional techniques in microbiology and related fields conventional in the art.

[0042] Example 1

[0043] 1. Prepare the enrichment medium for degradation:

[0044] (NH4)2SO4 2 g / L, sodium citrate 5 g / L, K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, NaCl 6 g / L, make up to 1 L with deionized water.

[0045] 2. The lake sludge samples were subjected to tolerance acclimation in the above-mentioned high-concentration enrichment medium. Each generation of acclimation took 3 days, and a total of five generations of acclimation culture were carried out to obtain a mixed bacterial solution. The culture temperature was kept constant at 35°C.

[0046] 3. Prepare the separation medium for degradation:

[0047] (NH4)2SO42g / L, sodium citrate 5g / L, K2HPO41.5 g / L, KH2PO40.5 g / L, MgSO4·7H2O 0.2g / L, NaCl 6g / L, agar powder 18g / L, dilute to 1L with deionized water;

[0048] 4. Inoculate the above bacterial liquid onto the separation medium, and separate single colonies from the liquid sample that has tolerated five generations of domestication. Use the dilution and spreading method, incubate at a constant temperature of 37°C in a constant temperature incubator for 48 hours, pick typical colonies, and streak them multiple times until single colonies are formed.

[0049] 5. Strain preservation:

[0050] Pick a single colony into LB liquid medium and culture it in a shaking incubator at 180 rpm and 37°C for 12 h. Mix the bacterial solution with 50% glycerol at a volume ratio of 1:1 to a final glycerol concentration of 25% and store it in a -80°C refrigerator.

[0051] Example 2

[0052] Based on the Castellaniella ginsengisoli IURM H19 obtained in Example 1, this example analyzes the morphological characteristics of Castellaniella ginsengisoli IURM H19.

[0053] 1. After single staining with crystal violet, the bacterial sample appears light purple on the slide and is observed under a microscope. The bacteria appear irregular rod-shaped under the microscope. Figure 1 .

[0054] 2. Morphological identification: After Castellaniella ginsengisoli IURM H19 was streaked on LB solid medium and grown for 36 hours, irregular colonies were formed with a smooth and moist surface, light milky white color, and no protrusions. Figure 2 .

[0055] Example 3

[0056] Based on the Castellaniella ginsengisoli IURM-H19 cultured in Example 1.

[0057] 1. DNA Sequencing

[0058] (1) DNA extraction: 2 mL of bacterial culture solution cultured to the logarithmic growth phase was concentrated and placed in a cell disruptor to disrupt the cell wall, and the DNA after cell wall disruption was collected.

[0059] (2) The extracted DNA samples are amplified using universal primers.

[0060] The PCR reaction system was as follows: 0.3 μL of template DNA, 15 μL of Taq enzyme mixture, 0.75 μL of forward primer, 0.75 μL of reverse primer, and sterile deionized water to 30 μL.

[0061] PCR amplification conditions: pre-deformation at 98°C for 7 min, followed by denaturation at 98°C for 15 s, amplification at 50-55°C for 15 s, and extension at 72°C for 40 s. After 33 cycles, the obtained fragments were used for sequencing.

[0062] Forward primer: 5'-GAGCGGATAACAATTTCACACAGG-3' (SEQ ID NO. 1); reverse primer: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' (SEQ ID NO. 2).

[0063] (3) PCR electrophoresis:

[0064] Observe the electrophoresis using 1.5% agarose gel in 1×TAE, 150V, 100mA, 20min environment. The results are as follows Figure 3 .

[0065] (4) Bidirectional sequencing: The amplified product was sent to Wuxi Saisuofei Biotechnology Co., Ltd. for first-generation bidirectional sequencing.

[0066] (5) Construction of phylogenetic tree:

[0067] The 16s rDNA sequences obtained by gene sequencing were compared with the NCBI database using Blast, and the 16s rDNA sequences with higher consistency were selected. The sequences were multiple aligned using MEGA software and a phylogenetic tree was constructed. Figure 4 , it was found that Castellaniella ginsengisoli had the highest sequence homology with a similarity of 98.87%, thus completing the identification of the obtained strain.

[0068] Example 4 Determination of ammonia nitrogen degradation efficiency

[0069] 1. Selection of detection method:

[0070] The phenol-sodium hypochlorite colorimetric method is used to determine the ammonia nitrogen concentration. Under alkaline conditions, ammonia is oxidized by sodium hypochlorite to generate chloramine and phenol, which react with sodium nitrosoferrocyanide to generate blue indophenol. Within a certain range, the color depth is proportional to the ammonia nitrogen content, and the absorption peak wavelength is 630nm.

[0071] 2. Drawing of standard curve: Weigh 0.6607g of ammonium sulfate that has been dried at 100℃ for 24h, add a small amount of water to dissolve it and then dilute it to a 100mL volumetric flask to obtain a 100mmol / L ammonium stock solution. Dilute the stock solution to prepare five standard ammonium solutions with different concentration gradients of 1.0, 2.0, 3.0, 4.0, and 5.0mmol / L. Add 50μL of standard ammonium solution to a test tube, use distilled water as a blank, add 2.5mL of phenol solution, shake well, add 2mL of sodium hypochlorite solution, mix again, and color in a 95℃ water bath for 5min. Take out and cool, then add 500μL to each well of a 48-well plate. Measure the absorbance value at a wavelength of 630nm in the fully automatic growth curve analyzer by colorimetry. Use the concentration of the standard ammonium solution as the horizontal coordinate and the absorbance value as the vertical coordinate to draw the ammonium standard curve as shown below: Figure 5 .

[0072] 3. Degradation efficiency test: After 36 hours of cultivation, samples were taken to determine the ammonia nitrogen concentration C. The ammonia nitrogen degradation rate was expressed by the formula:

[0073] Y(%)=100×(C0-C) / C0

[0074] The initial concentration of ammonia nitrogen is 300 mg / L. The dynamic curve of ammonia nitrogen degradation is as follows: Figure 6 As shown, the results show that the ammonia nitrogen concentration in the solution tends to decrease with the reaction time, and the downward trend gradually decreases, and the ammonia nitrogen concentration drops from 300 mg / L to 120 mg / L.

[0075] Example 5

[0076] Based on the Castellaniella ginsengisoli IURM-H19 cultured in Example 1, this example explores the effect of pH conditions on its degradation rate.

[0077] The inoculum was inoculated with 1% of the culture medium with an ammonia nitrogen concentration of 300 mg / L. The pH value of the culture medium was controlled to be maintained at 6.0, 7.0, and 8.0, respectively. The shaking speed was set to 180 rpm and the temperature was 35°C. Each group was repeated 3 times. After 36 hours, samples were taken to determine the residual ammonia nitrogen concentration in the culture medium and the ammonia nitrogen degradation rate was calculated.

[0078] like Figure 7 The degradation rate of ammonia nitrogen by the strain increased first and then decreased with the increase of pH. When the pH value was 7.0, the degradation rate of ammonia nitrogen by the strain reached 62.09%.

[0079] Example 6

[0080] Based on the Castellaniella ginsengisoli IURM-H19 cultured in Example 1, this example explores the effect of temperature conditions on its degradation rate.

[0081] The inoculum was inoculated at a rate of 1% into MSM culture medium with an ammonia nitrogen concentration of 300 mg / L. The shaking speed was set at 180 rpm and the culture temperatures were set at 25°C, 30°C, 35°C, and 40°C, respectively. Three parallels were set for each group. After 36 hours, samples were taken to determine the ammonia nitrogen concentration in the fermentation broth and the ammonia nitrogen degradation rate was calculated.

[0082] like Figure 8 The degradation rate of ammonia nitrogen by the strain increased first and then decreased with the increase of temperature. When the temperature was 30℃, the degradation rate of ammonia nitrogen by the strain reached a maximum of 60.08%.

[0083] Example 7

[0084] Based on the Castellaniella ginsengisoli IURM H19 cultured in Example 1, this example explores the effect of erythromycin on the removal rate of chemical oxygen demand (COD) in sewage.

[0085] After the pig manure sewage was diluted to a COD concentration of 500 mg / L, the diluted pig manure sewage sample was used as the control group, and erythromycin with a concentration of 0.1 mg / L was added to the diluted sample as the experimental group. The inoculation amount was 1% and inoculated into the pig manure sewage of the experimental group and the control group respectively. The shaking table speed was set to 180 rpm and the temperature was set to 35°C. Sampling was performed every 12 hours, and each group was repeated 3 times.

[0086] Dilute the pig manure wastewater sample to be tested to the range of 20-1000 mg / L, take 2.5 mL of the diluted sample into the digestion tube, add 0.7 mL of LH-D-100 reagent (oxidant) and 4.8 mL of LH-E-100 reagent (catalyst), cover the lid and put it into the digestion hole of the digestion instrument, cover it with a spray shield, and digest it at 165 ° C for 10 minutes. After the digestion is completed, cool it in the air for 2 minutes, add 2.5 ml of distilled water to each digestion tube and shake it well, and put the digestion tube into the water bath cooling tank to cool for 2 minutes. Unscrew the digestion tube cover, take a sample and measure the absorbance at 610 nm, use 0 mg / L COD as a reference to zero, and measure the absorbance of each sample. The COD concentration of pig manure wastewater is obtained according to the equation, and the actual COD concentration of the pig manure wastewater sample is calculated by multiplying it by the dilution multiple, and then the COD degradation rate is calculated.

[0087] like Fig. 9 After 60h of reaction time, the COD concentration in the experimental group decreased from 500mg / L to 150.08mg / L, with a degradation rate of 69.98%. The COD concentration in the control group decreased from 500mg / L to 131.23mg / L, with a degradation rate of 73.75%. It is expected that erythromycin inhibited the degradation of COD by bacteria.

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Castellaniella ginsengisoli IURM H19 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 23, 2024, with the deposit number CGMCC No.29727, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial agent, characterized in that it contains the above-mentioned Castellaniella ginseng.

3. Use of the Castellania ginseng described in claim 1 or the bacterial agent described in claim 2 in removing ammonia nitrogen from waste liquid.

4. The use according to claim 3, characterized in that: The Castellania ginseng or the bacterial agent according to claim 2 is added to the wastewater containing ammonia nitrogen to carry out a reaction.

5. The use according to claim 4, characterized in that: The OD600 value of the G. ginseng strain added to the wastewater was at least 0.

59.

6. The use according to claim 4, characterized in that: The reaction is carried out at 25-40° C., 180-220 rpm, and pH 6-8 for 0.5 h to 72 h.

7. The use according to claim 4, characterized in that: The reaction is carried out at 30-35°C, 180 rpm and pH 7 for 36 hours.

8. The use according to claim 4, characterized in that: The ammonia nitrogen concentration in the wastewater is 300-500 mg / L.

9. The use according to claim 4, characterized in that: The waste water is waste water from the production process of erythromycin.

10. Use of the Gastrocastella ginseng described in claim 1 in preparing a product for removing ammonia nitrogen from wastewater.

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