Acinetobacter YYD-3 and application

Abstract

The invention relates to the technical field of microorganisms, in particular to acinetobacter YYD-3 and application of the acinetobacter YYD-3, and the preservation number of the acinetobacter YYD-3 is CGMCC (China General Microbiological Culture Collection Center) No.29528. The acinetobacter YYD-3 provided by the invention shows strong adaptability and efficient denitrification capacity to different initial ammonia concentrations, has wide pH value adaptability, and can realize efficient denitrification of sewage in neutral, slightly alkaline and alkaline environments; in addition, the acinetobacter YYD-3 has certain salinity tolerance, and is suitable for being applied to treatment of various salt-containing wastewater. Compared with the prior art, the acinetobacter YYD-3 can realize one-stop efficient removal of ammonia nitrogen in a medium and low temperature environment, especially can realize long-time colonization in activated sludge, and remarkably improves the ammonia nitrogen removal capability of a sewage treatment system.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a kind of Acinetobacter YYD-3 and application thereof. Background Art

[0002] Ammonia nitrogen pollution brings great harm to the environment and ecosystem, causing water pollution, affecting the health of aquatic organisms, exacerbating eutrophication problems, and possibly threatening human health. Biological methods are widely used due to their economical, efficient, and environmentally friendly advantages.

[0003] Biological methods include two categories: phytoremediation and microbial treatment. Phytoremediation is favored because of its environmental sustainability, but its treatment capacity is limited. In contrast, microbial treatment can remove multiple nitrogen pollutants at the same time and avoid secondary pollution, but these factors have a significant impact on the efficiency of microbial denitrification. Studies have shown that, for example, in the range of 15°C to 35°C, Rhodococcus stink bug WM36 (CN 113699057 A) exhibits different degradation efficiencies in the removal of ammonia nitrogen. However, at lower temperatures (such as 15°C), the activity of strain WM36 is significantly inhibited, and this inhibition affects the colonization ability of the strain in the activated sludge system.

[0004] Cold-tolerant heterotrophic nitrification-aerobic denitrification bacteria can achieve the degradation and removal of ammonia nitrogen with almost no accumulation of nitrate and nitrite. However, this type of strain is easily affected by water environmental factors (such as temperature, pH, C / N ratio and ammonia nitrogen concentration) and has poor colonization ability in suspended water treatment systems; therefore, the existing studies on this type of strain are mostly limited to separation and purification and the study of denitrification characteristics under low temperature environments, lacking practical application strains.

[0005] Especially in the low-temperature sewage treatment in the northern cold regions in winter, the activity of activated sludge decreases at low temperatures, and low temperatures inhibit its activity and metabolic rate, which leads to changes in the microbial community, ultimately affecting the sewage treatment capacity of the strain, resulting in poor organic matter removal and denitrification effects, and seriously affecting the water quality of sewage treatment effluent.

[0006] With the implementation of the nationwide large-scale sewage treatment plant upgrading and transformation action, sewage treatment plants in the northern cold regions have selected appropriate measures to improve the performance of sewage biological treatment systems, and water plants in various regions have carried out technical upgrades and transformations to adapt to the water quality treatment needs of low-temperature environments. Improvement measures include introducing low-temperature-adapted microbial strains, optimizing reaction conditions and adjusting treatment processes to improve water quality treatment efficiency and stability in cold seasons and ensure that effluent is safe and meets standards.

[0007] In view of this, finding cold-resistant microorganisms with strong colonization ability is crucial to improving the efficiency and environmental protection level of sewage treatment plants. Summary of the invention

[0008] The purpose of the present invention is to provide a kind of Acinetobacter with heterotrophic nitrification-aerobic denitrification (HNAD) denitrification performance under medium and low temperature environment, which can realize "one-stop" efficient removal of ammonia nitrogen under medium and low temperature environment, especially can realize long-term colonization in activated sludge, and significantly improve the removal capacity of ammonia nitrogen in sewage treatment system.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a kind of Acinetobacter sp. YYD-3, and its preservation number is CGMCC No. 29528. The 16SrRNA identification sequence is as follows, the strain belongs to the genus Acinetobacter, and is named Acinetobacter sp. YYD-3. It is deposited in the General Microbiological Center (CGMCC) of China Microbiological Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation time is January 8, 2024.

[0011] Acinetobacter YYD-3 is isolated from the activated sludge in the return sludge of the second sedimentation tank of the fourth sewage treatment plant in Qinhuangdao City, Hebei Province. It should be noted that: after identification, the Acinetobacter YYD-3 of the present invention is a new Acinetobacter strain. Since the 16S sequence of the Acinetobacter YY D-3 of the present invention is closest to Acinetobacter guillouiae in the Acinetobacter strains of the prior art, the similarity is 98%, and the classification name suggested in the deposit certificate is temporarily written as Acinetobacter guillouiae. After investigation, no prior art reports of similar species separated from the activated sludge environment are found.

[0012] The present invention also provides an Acinetobacter YYD-3 preparation, which contains Acinetobacter YYD-3 with a preservation number of CGMCC No. 29528 and is a solid or liquid bacterial preparation.

[0013] The present invention also provides an application of Acinetobacter YYD-3 or an Acinetobacter YYD-3 preparation in removing ammonia nitrogen from sewage.

[0014] The present invention also provides an application of Acinetobacter YYD-3 or an Acinetobacter YYD-3 preparation in removing ammonia nitrogen from sludge.

[0015] Compared with the prior art, the Acinetobacter YYD-3 provided by the present invention exhibits strong adaptability and efficient denitrification ability to different initial ammonia nitrogen concentrations; at the same time, it has the ability to adapt to a wider pH value and can achieve efficient denitrification of sewage in neutral, alkaline and alkaline environments; in addition, Acinetobacter YYD-3 has a certain salinity tolerance and is suitable for the treatment of various types of saline wastewater.

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is the phylogenetic tree of Acinetobacter YYD-3 in the present invention.

[0019] Figure 2 This is a graph showing the denitrification performance of Acinetobacter YYD-3 in the present invention under different initial ammonia nitrogen environments at 10°C.

[0020] Figure 3 This is a graph showing the denitrification performance of Acinetobacter YYD-3 in the present invention under different pH environments at 10°C.

[0021] Figure 4 This is a graph showing the denitrification performance of Acinetobacter YYD-3 in the present invention under different C / N environments at 10°C.

[0022] Figure 5 This is a "one-stop" denitrification performance diagram of the Acinetobacter YYD-3 in the present invention under different salinity environments at 10°C.

[0023] Figure 6 This is a graph showing the denitrification performance of Acinetobacter YYD-3 added to activated sludge in the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0025] Isolation, screening and identification of strains Material preparation: The activated sludge in the return sludge of the secondary sedimentation tank of the Fourth Sewage Treatment Plant in Qinhuangdao City, Hebei Province was collected as a sample.

[0026] (1) Enrichment medium: used to enrich heterotrophic nitrifying aerobic denitrifying bacteria, the formula is 10g / L peptone, 5g / L yeast powder, 4.37g / L Na 2 HPO 4 12H 2 O, 1.83 g / L NaH 2 PO 4 ·2H 2 O, 10g / L NaCl, pH range 7-8. Enrichment medium was used to enrich bacteria at 30°C, 120rpm.

[0027] (2) Basic nitrification medium: used for bacterial screening experiments, shake flask batch culture experiments, etc. Its formula is 0.382g / L NH 4 Cl, 3.418 g / L CH 3 COONa, 4.37 g / L Na 2 HPO4·12H 2 O, 1.83 g / L NaH 2 PO 4 ·2H 2 O, 4g / LNaCl and 3mL / L trace elements.

[0028] (3) Basic nitrification solid culture medium composition: Add 2g agar powder to every 100mL basic nitrification culture medium. After high-pressure sterilization, cool the culture medium to about 50°C and pour it into a sterilized plate under aseptic operation. After the liquid in the plate condenses, a solid culture medium plate can be obtained. Before using the solid culture medium plate, it should be inverted for a period of time to ensure that no foreign bacteria enter the solid culture medium.

[0029] (4) Trace element ingredients per liter include: 3g MgSO 4 7H 2 O, 3.36 g MnSO 4 ·H 2 O, 3gZnSO 4 7H 2 O, 1.12 g H 3 BO 3 , 0.3 g FeSO 4 7H 2 O, 0.6 g CaCl 2 .

[0030] Experimental instruments and equipment: high-pressure steam sterilizer (DSX-24L-1), clean bench (SW-CJ-1FD), constant temperature shaking incubator (FS-70B), biochemical incubator (GDN-260), pH meter (PHS-25), UV-visible spectrophotometer (T6).

[0031] Using the activated sludge sample from the Fourth Sewage Treatment Plant in Qinhuangdao, Hebei Province, the bacterial flocs in the activated sludge were first broken up and left to stand for 10 minutes to obtain a bacterial suspension. Under a sterile environment, 1 mL of the bacterial suspension was inoculated into the basic nitrification medium and screened in a shaker with the parameters set to 30°C and a speed of 120 rpm; after 48 hours, another 1 mL of the bacterial suspension was taken from the basic nitrification medium to the new basic nitrification medium, and the screening process was repeated 3-4 times; finally, at 30°C and 120 rpm, the bacterial culture fluid was inoculated into the enrichment medium for enrichment for 24 to 48 hours to obtain an enriched primary screening culture fluid. Prepare 6 90 mL sterile triangular flasks and add 10 mL of the primary screening culture fluid to each bottle. Next, gradually dilute the primary screening culture fluid to prepare 10 -1 ~10 -6 Each dilution level of bacterial solution was evenly spread on a culture dish containing a basic nitrified solid culture medium using a spreading rod, and three culture dishes were spread in parallel for each dilution gradient. Then, these culture dishes were inverted and placed in a biochemical incubator and cultured at 10°C for more than 100 hours until colonies were clearly seen in the solid culture medium.

[0032] The colonies vary in size, shape and color. Under sterile conditions, single colonies were inoculated into culture bottles containing basic nitrification liquid medium. In a constant temperature shaker, the culture was continued at 10°C and 120 rpm for 48 hours. After the culture was completed, the cultured bacterial liquid was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected for determination of ammonia nitrogen removal and OD 600value. After multiple separations and purifications, the strain that showed the best ammonia nitrogen removal effect was selected as the target strain for ammonia nitrogen removal under low temperature conditions. Multiple coatings were performed to verify whether the pure bacterial liquid screened had consistent morphology on the plate culture medium and whether abnormal bacteria were mixed to ensure that the isolated strain was a single strain. Finally, the pure strain was inoculated onto the enrichment culture medium and refrigerated at 4°C for later use. The target strain was enriched and cultured in a constant temperature shaker at 30°C and 120rpm for 24h using the enrichment culture medium, and the bacteria were collected using a high-speed centrifuge (8000rpm, 10min). The genomic DNA of the strain was extracted using the reagents and instruments in the bacterial DNA kit, and the DNA fragment was amplified using PCR technology (polymerase chain reaction) using the obtained DNA template to obtain the 16S rRNA sequence of the strain. The primers for 16S rRNA are universal primers 27F and 1492R. Analysis of the 16SrRNA gene sequencing results showed that the effective length of the 16S rRNA gene sequence of the target strain was 1417bp, as follows:

[0033] The 16S rRNA sequence of Acinetobacter YYD-3 is shown in SEQ ID No. 1:

[0034] SEQ ID No.1:

[0035]

[0036] The BLAST program was used to obtain homologous bacterial genus-related sequences from the GenBank data National Center for Biotechnology Information (NCBI). The 16S rRNA sequence of strain YYD-3 was compared in the NCBI nucleotide database, and it was found that the homology between strain YYD-3 and Acinetobacter guillouiae was 98%. After multiple sequence alignment using MEGA11 software, a neighbor-joining (NJ) phylogenetic tree was constructed based on the K-2-P (Kimura-2-parameter) model, with a Bootstrap test of 1000. The constructed phylogenetic tree is shown in Figure 1. Figure 1 As shown, strain YYD-3 is most closely related to Acinetobacter guillouiae, and it is inferred that strain YYD-3 belongs to Acinetobacter guillouiae. It is deposited in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Administration, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 29528.

[0037] Example 1

[0038] The denitrification effect and adaptability of strain YYD-3 under different initial ammonia nitrogen concentration environments were studied. Three different initial ammonia nitrogen concentration groups (50 mg / L, 100 mg / L and 200 mg / L) were designed to simulate the possible ammonia nitrogen concentration gradient in different water bodies or soils. During the experiment, the temperature was controlled at 10°C and the pH value was maintained at (7-8) neutral to weakly alkaline to ensure the stability and consistency of the growth environment of the strain. During the 72h experimental period, samples were collected regularly and the changes in the residual ammonia nitrogen concentration were analyzed. The experimental results are as follows Figure 2It was shown that when the initial ammonia nitrogen concentration was 50 mg / L, strain YYD-3 exhibited significant ammonia nitrogen removal ability, with a removal rate of 96% and a total nitrogen removal rate of 74%, with only 1.60 mg of ammonia nitrogen not being degraded. When the initial ammonia nitrogen concentration rose to 100 mg / L, the denitrification effect of the strain was still significant, with an ammonia nitrogen removal rate of 91% and a total nitrogen removal rate of 80.28%. However, when the initial ammonia nitrogen concentration was further increased to 200 mg / L, the ammonia nitrogen removal rate of strain YYD-3 was significantly reduced to 46%, and the total nitrogen removal rate was 40%. Despite this, the strain still successfully degraded 92 mg of ammonia nitrogen, showing that it still has a strong denitrification ability under high ammonia nitrogen concentration conditions. These results show that strain YYD-3 exhibits strong adaptability and efficient denitrification ability to different initial ammonia nitrogen concentrations, providing a theoretical basis and experimental data support for its application in environmental engineering such as urban sewage treatment and aquaculture wastewater treatment processes.

[0039] Example 2

[0040] pH value is one of the important environmental factors in bacterial growth and metabolism. In order to investigate the denitrification effect and adaptability of the strain in culture media with different pH values, this experiment used 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution to adjust the initial pH value of the nitrification medium to 5, 7, 9, 10 and 11, respectively. After the activated strain YYD-3 was inoculated into the basic nitrification medium, it was cultured at 10°C and 120rpm. At different time points in the culture (0h, 12h, 24h, 36h, 42h, 48h, 60h and 72h), samples were collected and the concentrations of ammonia nitrogen and total nitrogen in the supernatant of the water samples were determined. According to Figure 3 The data showed that after 72 hours of cultivation, under the condition of pH = 10, strain YYD-3 showed the highest ammonia nitrogen removal rate (97.96%) and total nitrogen removal rate (88.18%). After 48 hours of cultivation, under neutral-alkaline conditions (pH = 7-10), the ammonia nitrogen removal rate and total nitrogen rate increased significantly. In summary, strain YYD-3 has the ability to adapt to a wide pH value and can achieve efficient denitrification of sewage in neutral, alkaline and alkaline environments.

[0041] Example 3

[0042] The ratio of carbon source and nitrogen source has an important influence on the growth and metabolism of denitrifying bacteria. When the carbon-nitrogen ratio is too low, the biomass and denitrification efficiency of denitrifying organisms will decrease; while a high carbon-nitrogen ratio may lead to an increase in the microbial load, inhibit the growth of microorganisms, and at the same time, lead to a waste of carbon source resources. In order to study the denitrification effect and adaptability of strain YYD-3 at different carbon-nitrogen ratios. In this experiment, urban domestic sewage was used as the target water body, the ammonia nitrogen concentration was 100 mg / L, and the carbon-nitrogen ratio was set to 5, 10 and 20. The strain YYD-3 was cultured at 10°C, and samples were taken at different time points (0h, 12h, 24h, 36h, 42h, 48h, 60h and 72h) to determine the ammonia nitrogen and total nitrogen concentrations in the supernatant. According to Figure 3 The results showed that after culturing at 10°C for 72 hours, the removal efficiency of ammonia nitrogen by strain YYD-3 increased significantly with the increase of carbon-nitrogen ratio. After 60 hours, when the carbon-nitrogen ratio was 5, the removal rates of ammonia nitrogen and total nitrogen by strain YYD-3 quickly reached 67.49% and 60.75%, and then the ammonia nitrogen degradation rate decreased, which was lower than the ammonia nitrogen removal rates of 68.45%, 90.68% and 94.90% and the total nitrogen removal rates of 62.13%, 80.28% and 94.60% when the carbon-nitrogen ratio was 10 and 20.

[0043] Example 4

[0044] Salinity has a significant impact on the growth, metabolic pathways and ecological functions of microorganisms. Microorganisms that can survive in low, medium and high salt environments have important application potential in environmental engineering and biotechnology. In order to verify the denitrification effect and adaptability of strain YYD-3 under different salt concentration conditions, the culture medium salinity was set to 1g / L, 2g / L, 4g / L, 6g / L, 8g / L and 15g / L under the conditions of ammonia nitrogen concentration of 100mg / L and pH value of 7. Figure 5 It can be seen that the removal effect of ammonia nitrogen by strain YYD-3 under different salinity conditions is basically similar, and with the increase of salinity, the ammonia nitrogen removal rate is 93.71% (1g / L)>90.87% (2g / L)>90.40% (4g / L)>89.05% (6g / L)>78.51% (8g / L)>76.31% (15g / L), and the total nitrogen removal rate decreases from 86.87% to 62.52%. This shows that strain YYD-3 has a certain degree of salinity tolerance and is suitable for the treatment of various types of saline wastewater.

[0045] Example 5

[0046] Activated sludge in sewage treatment plants is inhibited by low temperature in winter. This study verifies whether strain YYD-3 can colonize in activated sludge and improve the low-temperature denitrification effect of activated sludge by adding it to activated sludge. Activated sludge samples collected from the biological aeration tank of Qinhuangdao No. 3 Sewage Treatment Plant were transferred to BNM culture medium after static sedimentation, and strain YYD-3 was added at a volume ratio of 3%. The shaker temperature and speed were set to 10°C and 120rpm respectively, and the culture was carried out at low temperature for 24 hours. An experimental group and a control group without the addition of the strain were set. The results are shown in Figure 6 As shown, the low-temperature degradation rate of ammonia nitrogen in the experimental group with added YYD-3 was 65.26%, which was significantly higher than 36.11% in the control group. The removal effect of ammonia nitrogen in the activated sludge with added bacterial agent was about 1.8 times that of the control group. This shows that the strain can colonize in activated sludge and improve the low-temperature denitrification performance of activated sludge.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

Claims

1. A kind of Acinetobacter YYD-3, whose deposit number is CGMCC No.29528.

2. An Acinetobacter YYD-3 preparation, which contains Acinetobacter YYD-3 with a deposit number of CGMCC No. 29528, and is a solid or liquid bacterial preparation.

3. Use of the Acinetobacter YYD-3 described in claim 1 or the Acinetobacter YYD-3 preparation described in claim 2 in removing ammonia nitrogen from sewage.

4. Use of the Acinetobacter YYD-3 described in claim 1 or the Acinetobacter YYD-3 preparation described in claim 2 in removing ammonia nitrogen from activated sludge.

5. Use of the Acinetobacter YYD-3 described in claim 1 or the Acinetobacter YYD-3 preparation described in claim 2 for colonization in activated sludge.

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