Strain for removing ammonia nitrogen and application thereof

Abstract

The application discloses a ginseng soil Castellaniella strain and application, the ginseng soil Castellanniella strain is named IURM-H19, is preserved in China Microbial Culture Collection Center, the preservation number is CGMCC No.29727, and the preservation date is January 23, 2024; the ginseng soil Castellanniella strain IURM-H19 of the application can degrade ammonia nitrogen in sewage, and also has good degradation effect on COD in sewage; the ginseng soil Castellanniella strain IURM-H19 of the application can develop corresponding biological agents, is used for the degradation of ammonia nitrogen and COD in environmental sewage, prevents water body eutrophication, and reduces its harmful effect on human and aquatic organisms.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation technology, specifically relating to a strain that removes ammonia nitrogen and its application. Background Technology

[0002] Whether in a high-concentration or low-concentration state, excessive ammonia nitrogen content in a water system will cause varying degrees of harm, mainly as follows: (1) Ammonia nitrogen will consume a large amount of dissolved oxygen in the aquatic environment, causing water deterioration, and in severe cases, even turning black and smelly, resulting in a decline in water quality and posing a threat to organisms living in it; (2) Ammonia nitrogen can react with substances in the water system to generate secondary pollutants, causing secondary pollution, which will increase the treatment cost; (3) Too much nitrogen in the water will lead to eutrophication, causing an increase in the number of algae and accelerating the rate of water deterioration; (4) Nitrite and nitrate nitrogen substances in the water will cause significant harm to humans and aquatic organisms. Some of these substances can cause cancer, mutation, and malformation when accumulated in large quantities. Furthermore, excessive ammonia nitrogen content in water bodies can also lead to serious incidents such as poisoning of humans and animals and "implicate" related water bodies. Based on these factors, the treatment of ammonia nitrogen in water bodies is not only a hot topic, but also constitutes a necessary link in environmental protection.

[0003] In recent years, nitrogen compounds have continued to accumulate in the environment, especially affecting water pollution. When nitrogen compounds enter water bodies and undergo chemical changes to transform into more stable secondary pollutants, they can 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 constantly increasing, and they are gradually realizing the importance of treating nitrogen-containing wastewater, especially ammonia-containing wastewater.

[0004] By utilizing microbial methods to degrade nitrogenous substances in wastewater and exploring the metabolic patterns of bacterial strains, this study provides valuable reference for the engineering applications of these strains and is of great significance for protecting the water environment and mitigating the water resource crisis.

[0005] Currently, commonly used methods for removing ammonia nitrogen mainly include physical precipitation, chemical precipitation, and biological methods. Biological methods for degrading ammonia nitrogen have significant advantages over mainstream methods such as physicochemical methods, primarily in terms of high cost-effectiveness, environmental friendliness, stable treatment results, strong resource recovery capacity, high treatment capacity, high sustainability, good flexibility, strong adaptability, mature technology, and wide treatment range. New nitrogen removal technologies such as short-cut nitrification-denitrification and anaerobic ammonia oxidation have strict process requirements in practical applications, especially regarding dissolved oxygen. Therefore, obtaining a strain of bacteria that effectively treats ammonia nitrogen in wastewater and has a short degradation cycle remains a problem to be solved. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

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

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a ginseng-derived *Castella ginseng*.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Castella ginsengisoli IURM H19, was deposited on January 23, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29727, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] Another objective of this invention is to overcome the shortcomings of the prior art and provide a microbial inoculant containing *Castanone ginseng*.

[0011] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of *Castanone ginseng* or a bacterial agent containing *Castanone ginseng* in the removal of ammonia nitrogen from waste liquid.

[0012] In a preferred embodiment of the application described in this invention, *Castanone ginseng* or a bacterial agent containing *Castanone ginseng* is added to wastewater containing ammonia nitrogen to carry out a reaction.

[0013] As a preferred embodiment of the application described in this invention, the OD600 value of the *Castanone ginseng* added to the wastewater is at least 0.59.

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

[0015] As a preferred embodiment of the application described in this invention, the reaction is carried out at 30–35°C, 180 rpm, and pH 7 for 36 h.

[0016] In a preferred embodiment of the application described in this invention, the ammonia nitrogen concentration in the wastewater is 300–500 mg / L.

[0017] In a preferred embodiment of the application described in this invention, the wastewater is wastewater from the erythromycin production process.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of *Castanone ginseng* in the preparation of products for removing ammonia nitrogen from wastewater.

[0019] Beneficial effects of this invention:

[0020] 1. This invention is the first to discover a type of *Castella ginseng* that degrades ammonia nitrogen using a biological treatment method. It has the advantages of high deodorization, high biodegradation efficiency, no secondary pollution, and low cost.

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

[0022] 3. The *Castanone ginseng* IURM-H19 of the present invention can be used to develop corresponding biological agents for the degradation of ammonia nitrogen and COD in environmental wastewater, preventing eutrophication of water bodies and reducing their harmful effects on humans and aquatic organisms. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments 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. Wherein:

[0024] Figure 1 This is a crystal violet monostained 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 Embodiment 3 of the present invention.

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

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

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

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

[0032] Figure 9 The COD removal efficiency of IURM H19 on pig manure wastewater in Example 7 of this invention. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "an 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 phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

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

[0037] Table 1

[0038]

[0039] Crystal violet staining:

[0040] First, use a pipette to draw an appropriate amount of bacterial suspension onto a glass slide, gently spread the suspension, and dry it over a low flame. Next, when a bacterial film forms on the slide, add 1-2 drops of crystal violet for staining. After staining for 1-2 minutes, rinse off the stain with a slow stream of water, and then blot away any remaining moisture with absorbent paper. Finally, examine the slide under a microscope, first finding the field of view under low magnification, then switching to high magnification to observe the specific morphological characteristics of the bacterial strain.

[0041] Unless otherwise stated, the experimental methods disclosed in this invention all employ conventional techniques in the fields of microbiology and related fields.

[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, and dilute to 1 L with deionized water.

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

[0046] 3. Prepare the isolation culture medium for degradation:

[0047] (NH4)2SO4 2g / L, sodium citrate 5g / L, K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2g / L, NaCl 6g / L, agar powder 18g / L, and dilute to 1L with deionized water;

[0048] 4. Inoculate the above bacterial suspension onto an isolation medium and isolate single colonies from liquid samples that have tolerated five generations of acclimatization. Use the dilution plating method and incubate at 37°C for 48 hours. Pick typical colonies and streak them multiple times until single colonies are formed.

[0049] 5. Strain preservation:

[0050] Single colonies were picked and placed into LB liquid medium and incubated at 180 rpm and 37°C for 12 h on a shaker. The bacterial culture was then mixed with 50% glycerol at a volume ratio of 1:1 to achieve a final glycerol concentration of 25%, and stored at -80°C.

[0051] Example 2

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

[0053] 1. After single staining with crystal violet, the bacterial sample appears pale purple on the slide. When observed under a microscope, the bacteria appear as irregular rod-shaped structures. Figure 1 .

[0054] 2. Morphological identification: After streaking Castella ginsengisoli IURM H19 onto LB solid medium and growing for 36 hours, it formed irregularly shaped colonies with a smooth, moist surface and a light milky-white color, without any raised areas. Figure 2 .

[0055] Example 3

[0056] The ginseng-derived Castellaniella ginsengisoli IURM-H19 was cultured based on Example 1.

[0057] 1. DNA sequencing

[0058] (1) DNA extraction: Take 2 mL of bacterial culture that has been cultured to the logarithmic growth phase, concentrate it, and place it in a cell disruptor to disrupt the cell wall. Collect the DNA after disruption.

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

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

[0061] PCR amplification conditions: pre-denaturation at 98℃ for 7 min, followed by denaturation at 98℃ for 15 s, amplification at 50-55℃ for 15 s, extension at 72℃ for 40 s, and 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] The results were observed using a 1.5% agarose gel electrophoresis at 1×TAE, 150V, 100mA, for 20 min. Figure 3 .

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

[0066] (5) Construction of the phylogenetic tree:

[0067] The 16S rDNA sequences obtained from gene sequencing were compared with the NCBI database using BLAST. Sequences with high consistency were selected, and multiple sequence alignment was performed using MEGA software to construct a phylogenetic tree. Figure 4 The results showed that Castellaniella ginsengisoli had the highest sequence homology with Castellaniella ginsengisoli, 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] Ammonia nitrogen concentration was determined by the phenol-sodium hypochlorite colorimetric method. Under alkaline conditions, chloramine, which is generated by the oxidation of ammonia by sodium hypochlorite, reacts with phenol to form blue indophenol under the catalysis of sodium nitrosoferricyanide. Within a certain range, the intensity of the color is directly proportional to the ammonia nitrogen content, and the absorption peak wavelength is 630 nm.

[0071] 2. Standard Curve Construction: Weigh 0.6607 g of ammonium sulfate dried at 100℃ for 24 h, dissolve it in a small amount of water, and dilute to volume in a 100 mL volumetric flask to obtain a 100 mmol / L ammonium stock solution. Dilute the stock solution to prepare five different concentration gradients of ammonium standard solutions: 1.0, 2.0, 3.0, 4.0, and 5.0 mmol / L. Add 50 μL of the standard ammonium solution to a test tube, using distilled water as a blank, add 2.5 mL of phenol solution, shake well, add 2 mL of sodium hypochlorite solution, mix again, and incubate at 95℃ for 5 min. After cooling, add 500 μL of the solution to each well of a 48-well plate and measure the absorbance at 630 nm using an automated growth curve analyzer. Plot the ammonium standard curve with the concentration of the standard ammonium solution on the x-axis and the absorbance on the y-axis as shown in the figure. 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 dynamic curve of ammonia nitrogen degradation at an initial concentration of 300 mg / L is shown in the figure below. Figure 6 As shown, the results indicate that the ammonia nitrogen concentration in the solution decreases with the progress of the reaction, and the decreasing trend gradually slows down, with the ammonia nitrogen concentration decreasing 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 investigates the effect of pH conditions on its degradation rate.

[0077] Inoculate 1% of the culture medium into MSM medium with an ammonia nitrogen concentration of 300 mg / L. Maintain the pH of the medium at 6.0, 7.0, and 8.0, respectively. Incubate at 180 rpm and 35°C. Perform three replicates per group. After 36 hours, take samples to determine the residual ammonia nitrogen concentration in the medium and calculate the ammonia nitrogen degradation rate.

[0078] like Figure 7 The degradation rate of ammonia nitrogen by the strain first increased and then decreased with increasing pH, reaching a maximum of 62.09% when the pH value was 7.0.

[0079] Example 6

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

[0081] Inoculate 1% of the culture medium into MSM medium with an ammonia nitrogen concentration of 300 mg / L. With the shaker speed set to 180 rpm, the culture temperature was set to 25℃, 30℃, 35℃, and 40℃, with 3 replicates for each group. After 36 hours, samples were taken to measure the ammonia nitrogen concentration in the fermentation broth and calculate the ammonia nitrogen degradation rate.

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

[0083] Example 7

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

[0085] After diluting the original pig manure wastewater to a COD concentration of 500 mg / L, the diluted pig manure wastewater sample was used as the control group, and erythromycin at a concentration of 0.1 mg / L was added to the diluted sample as the experimental group. The inoculation amount was 1% and added to the pig manure wastewater of the experimental group and the control group respectively. The shaking speed was set to 180 rpm and the temperature was set to 35℃. Samples were taken every 12 hours, and each group was repeated 3 times.

[0086] Dilute the pig manure wastewater sample to be tested to a concentration range of 20–1000 mg / L. Take 2.5 mL of the diluted sample into a digestion tube, add 0.7 mL of LH-D-100 reagent (oxidant) and 4.8 mL of LH-E-100 reagent (catalyst), cap the tube, place it in the digestion port of the digester, cover with a blowout shield, and digest at 165℃ for 10 min. After digestion, cool in air for 2 min, add 2.5 mL of distilled water to each digestion tube and shake well. Place the digestion tube in a water bath for 2 min. Unscrew the digestion tube cap, take a sample, and measure the absorbance at 610 nm. Zero the tube with 0 mg / L COD as a reference and measure the absorbance of each sample. Calculate the COD concentration of the pig manure wastewater according to the equation, multiply by the dilution factor to calculate the actual COD concentration of the pig manure wastewater sample, and then calculate the COD degradation rate.

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

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Castella ginsengisoli IURM H19 was deposited on January 23, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29727, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A microbial inoculant, characterized in that it contains the aforementioned *Castelloniella ginseng*.

3. The application of the *Castelloniella ginseng* strain of claim 1 or the bacterial agent of claim 2 in the removal of ammonia nitrogen from waste liquid.

4. The application as described in claim 3, characterized in that: The *Castelloniella ginseng* or the bacterial agent described in claim 2 is added to wastewater containing ammonia nitrogen to carry out a reaction.

5. The application as described in claim 4, characterized in that: The OD600 value of *Castanone ginseng* added to the wastewater should be at least 0.

59.

6. The application as described in claim 4, characterized in that: The reaction is carried out at 25–40°C, 180–220 rpm, and pH 6–8 for 0.5–72 h.

7. The application as described in claim 4, characterized in that: The reaction was carried out at 30–35°C, 180 rpm, and pH 7 for 36 h.

8. The application as described in claim 4, characterized in that: The ammonia nitrogen concentration in the wastewater is 300–500 mg / L.

9. The application as described in claim 4, characterized in that: The wastewater is from the erythromycin production process.

10. The use of the *Castanella ginseng* strain according to claim 1 in the preparation of products for removing ammonia nitrogen from wastewater.

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