Aerobic denitrifying bacterium with zoogloea character and application thereof

Abstract

The invention provides an aerobic denitrifying bacterium with zoogloea character and application thereof, and belongs to the technical field of wastewater treatment, the aerobic denitrifying bacterium is specifically acinetobacter baumannii N-1 (Acinetobacter sp.N-1), the acinetobacter baumannii is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242568. The acinetobacter baumannii N-1 provided by the invention not only can perform wastewater denitrification through aerobic denitrification and has efficient denitrification capacity, but also can realize separation of sludge and purified water by forming zoogloea, so that the acinetobacter baumannii N-1 has good application potential in wastewater treatment by utilizing an activated sludge process.

Description

Technical Field

[0001] The present invention relates to the technical field of wastewater treatment, and in particular to a strain of aerobic denitrifying bacteria with floc characteristics. Acinetobacter sp.N-1 (Acinetobacter baumannii N-1) and its application. Background Art

[0002] Excessive nitrogen is one of the important factors that lead to the reduction of production of aquatic products such as fish and shrimp. With the rapid development of aquaculture, high-density integrated pond farming models often cause water pollution, especially serious excess of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water bodies, which have direct and serious toxic effects on farmed aquatic products, causing huge economic losses. At the same time, secondary intake of aquatic products by humans can cause physical harm or even death. Therefore, the management of nitrogen in aquaculture has received increasing attention.

[0003] Generally speaking, biological denitrification can meet the requirements of water purification and has the advantages of low price and easy treatment of secondary pollutants. Traditional biological denitrification is completed by aerobic nitrification and anaerobic denitrification. In 1980, Meiberg et al. first discovered that denitrification can also be achieved under aerobic conditions. Subsequently, Robertson et al. first isolated a strain of aerobic denitrifying bacteria. Thiosphaera pantotropha (Now known as Paracoccus pantotrophus ), is a type of microorganism that can still use nitrate as an electron acceptor to produce energy in the presence of oxygen and convert nitrate into nitrogen gas or nitrogen dioxide. This discovery provides a new idea for biological denitrification - heterotrophic nitrification-aerobic denitrification. Compared with anaerobic denitrifying bacteria, the advantage of aerobic denitrifying bacteria is that they have a certain tolerance to dissolved oxygen in the culture medium or environment, allowing ammoniation, nitrification and denitrification reactions to act simultaneously in the aerobic environment of wastewater. Subsequently, Robertson et al. reported aerobic denitrifying bacteria ( Paracoccus sp. ) and more and more types of aerobic denitrifying bacteria have been isolated from different environments. Currently known aerobic denitrifying bacteria are mainly Pseudomonas ( Pseudomonas ), Acinetobacter spp. Acinetobacter )、Alcaligenes spp. Alcaligenes ), Bacillus ( Bacillus ), Delftia ( Delftia ) and Halomonas ( Halomonas )wait.

[0004] The activated sludge process is a method of treating sewage through aerobic organisms. It was first invented by two British scientists, Edward Ardern and William T. Lockett, in 1914. It is a general term for various methods of treating pollutants suspended in water through wastewater organisms. Activated sludge is a general term for microbial communities and the organic and inorganic substances they are attached to. It can remove soluble, colloidal biodegradable organic matter, suspended solids that can be adsorbed by activated sludge, and some other substances from sewage, and can also remove some phosphorus and nitrogen. The activated sludge process mainly works through three types of bacteria: floc-forming bacteria, polyphosphate bacteria, and denitrifying bacteria. As the main biological denitrification method at present, the key process of the activated sludge process is to use floc-forming bacteria to settle under gravity to achieve the separation of sludge and clean water. Therefore, the development of microorganisms with both aerobic denitrification ability and floc-forming ability is of practical significance for wastewater treatment using the activated sludge process. However, there are very few reports of strains with both these two characteristics in the prior art. Summary of the invention

[0005] In view of this, the present invention aims to separate and select an aerobic denitrifying bacterium with floc characteristics from nature, so as to provide a new microbial material for the field of wastewater treatment.

[0006] The technical solution of the present invention is as follows: In the first aspect, the present invention provides an aerobic denitrifying bacterium having flocculent properties, specifically Acinetobacter baumannii N-1 ( Acinetobacter sp.N-1), which is deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M 20242568, the deposit date is November 15, 2024, and the deposit address is Wuhan University, Wuhan City, Hubei Province.

[0007] The aerobic denitrifying bacteria with flocculent characteristics provided by the present invention are isolated from the activated sludge of the anoxic tank of a sewage treatment plant in Wuhan. The strain has the highest homology similarity with Acinetobacter baumannii through molecular biological identification. At the same time, morphological characteristics research shows that the plate colony characteristics of the strain are milky yellow, round, smooth surface and neat edges. Microscopic observation and measurement show that the strain is rod-shaped, 1.41-1.91μm long and 0.75-1.22μm wide. It is a Gram-negative bacterium and is not easy to decolorize during Gram staining. Combined with molecular and morphological analysis, the strain is named Acinetobacter sp.N-1.

[0008] The present invention provides Acinetobacter sp.N-1 has both the ability to aerobic denitrification and the ability to form flocs, a feature not reported in previous studies on Acinetobacter baumannii. This may be due to the fact that the strain Acinetobactersp.N-1 comes from activated sludge. During the activated sludge cultivation process, gene mutation or horizontal gene transfer occurred, which gave it the characteristic of producing flocs.

[0009] In a second aspect, the present invention provides a microbial agent, wherein the microbial agent is Acinetobacter sp.N-1 is one of the active ingredients. It is understandable that the microbial agent may also include other aerobic nitrifying bacteria, denitrifying bacteria, floc-forming bacteria, polyphosphate bacteria, etc., to achieve synergistic denitrification and / or wastewater treatment.

[0010] In a third aspect, the present invention provides the use of the above-mentioned aerobic denitrifying bacteria and microbial agents having floc characteristics, which are at least any of the following: a) Application in wastewater treatment; b) Application in the preparation of activated sludge.

[0011] The experimental data of the present invention show that the aerobic denitrifying bacteria provided by the present invention have good denitrification ability, so they can be used to remove nitrogen from wastewater; at the same time, the aerobic denitrifying bacteria have the ability to form bacterial flocs, which is more conducive to constructing activated sludge, and then realizing wastewater denitrification treatment through the activated sludge method.

[0012] In a fourth aspect, the present invention provides a method for treating wastewater, specifically comprising: in wastewater, using Acinetobacter sp.N-1 is used for denitrification treatment; the wastewater includes but is not limited to aquaculture wastewater, urban domestic sewage, etc.

[0013] Preferably, in the above wastewater treatment method, the pH of the wastewater is 7-9, the temperature is 25-35°C, and the C / N is 5-15.

[0014] Impact on the present invention Acinetobacter The factors affecting the denitrification efficiency of sp.N-1 include, but are not limited to, carbon source, C / N, temperature, pH, DO, shaker speed, inoculation amount, etc. The experimental data show that pH, temperature, C / N have a significant impact on the denitrification efficiency of sp.N-1. Acinetobacter sp.N-1 has a great influence on the denitrification efficiency and application range. In one embodiment of the present invention, these three factors are selected for response surface analysis and the theoretical optimal conditions are obtained. A quadratic regression fitting is performed on them. After the quadratic regression fitting equation is obtained, the optimal theoretical conditions can be obtained by solving it. The optimized denitrification conditions are: pH = 8.339, culture temperature = 25.176 ° C, C / N = 13.737 (R 校正 ²=0.9852, P-value<0.0001, lack of fit term=0.3284>0.05), the predicted denitrification efficiency was 76.89%; further verified by parallel experiments under this condition, the denitrification efficiency was 74.36%±1.041%, and the experimental deviation was only 3.28%. The 3D analysis diagram and corresponding contour map constructed by the response surface method intuitively show the interaction between various factors. It can be seen that the application of the response surface method in denitrification optimization is a more convenient and feasible method; moreover, the present invention successfully used the strain Acinetobacter The total nitrogen removal rate of sp.N-1 increased from 51% to 74.357%, and the denitrification effect increased by 45.79%.

[0015] Preferably, in the above wastewater treatment method, the wastewater is treated by an activated sludge process.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention reports for the first time a baumannii strain that has both aerobic denitrification ability and the ability to form bacterial flocs. The strain can denitrify wastewater through aerobic denitrification and has a highly efficient denitrification ability, and can also separate sludge from clean water by forming bacterial flocs. Therefore, the strain has significant advantages in wastewater treatment using the activated sludge method, and has good application potential in the fields of wastewater treatment such as aquaculture wastewater and urban domestic sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The figure is a graph showing the determination results of total nitrogen and nitric nitrogen removal rates of different strains in Example 1 of the present invention; Figure 2 This is a diagram showing the morphological identification results of Acinetobacter baumannii N-1 in Example 1 of the present invention; Figure 3 is the phylogenetic tree of Acinetobacter baumannii N-1 in Example 1 of the present invention; Figure 4 The effect of the two-level interaction of three factors in Example 2 of the present invention on the removal of total nitrogen by Acinetobacter baumannii N-1, wherein (a) is the binary interaction of temperature and pH, (b) is the binary interaction of C / N and pH, and (c) is the binary interaction of C / N and temperature. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0021] Aquaculture technology is currently widely used in the artificial breeding of various economic aquatic organisms. The aquaculture wastewater generated by it needs to be treated promptly and effectively, and the activated sludge method has been widely used in the treatment of various types of urban sewage and wastewater. As is well known in the industry, one of the key factors affecting the treatment effect of the activated sludge method is the various bacteria in the sludge, among which bacteria with the ability to form flocs and bacteria with denitrification are essential bacterial species, but there are very few reports on strains with these two properties in the prior art. In order to provide a richer selection of bacteria for wastewater treatment by the activated sludge method, the present invention provides an aerobic denitrifying bacterium with floc properties, namely Acinetobacter sp.N-1.

[0022] Aerobic denitrifying bacteria are aerobic or facultative aerobic bacteria, which are less abundant in nature and difficult to separate from environmental microorganisms using general microbial screening methods. However, aerobic denitrifying bacteria are relatively abundant in activated sludge plants. Acinetobacter sp.N-1 was isolated from the activated sludge of the anoxic tank of a sewage treatment plant in Wuhan. Although existing reports have disclosed that some strains of Acinetobacter baumannii have aerobic denitrification capabilities, they do not form flocs. For example, Wang Jing et al. first reported on the growth characteristics and aerobic denitrification capabilities of Acinetobacter baumannii in 2016. The detailed morphological identification results of the strains were almost consistent with this experiment, but it did not form flocs. As reported by Yang Zidong, the Acinetobacter baumannii from cattle sources also did not form flocs. Existing studies generally believe that there is no obvious correlation between the biofilm-forming ability of Acinetobacter baumannii and its genotype, and the formation of biofilms may be regulated by other genetic or environmental factors. In addition, in the study of attempting to screen out aerobic denitrifying bacteria that can form flocs from activated sludge, the present invention isolated 18 strains of aerobic denitrifying bacteria, but only Acinetobacter sp.N-1 has the ability to form flocs.

[0023] The present invention provides Acinetobactersp.N-1, orthogonal experiments can be used to optimize the culture conditions of the strains at different levels, explore the optimal combination of strains to obtain denitrification performance under the influence of multiple factors, and thus provide a theoretical basis and practical feasibility for wastewater treatment.

[0024] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0025] The composition of the culture medium used in the following examples is as follows.

[0026] BTB culture medium: Na2HPO4·7H2O 7.9g / L, KH2PO41.0g / L, NH4Cl 0.3g / L, MgSO4·7H2O0.1g / L; sodium succinate 4.7g / L, KNO31.0g / L, NaNO20.5g / L, trace element solution 2.0mL / L, 1% bromothymol blue ethanol solution 1mL / L; pH is 6.8-7.0.

[0027] Denitrification enrichment medium: Na2HPO4·7H2O 7.9g / L, KH2PO41.5g / L, NH4Cl 0.3g / L, MgSO4·7H2O 0.1g / L, sodium succinate 4.7g / L, KNO31.0g / L, NaNO20.5g / L, trace element solution 2.0mL / L.

[0028] Denitrification assay culture medium: Na2HPO4·7H2O 7.9 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.1 g / L, sodium succinate 4.7 g / L, KNO3 1.0 g / L, trace element solution 2.0 mL / L.

[0029] R2A culture medium: 0.5 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L starch, 0.5 g / L acid hydrolyzed casein, 0.3 g / L sodium pyruvate, 0.3 g / L K2HPO4, 15 g / L agar (added for solid culture medium), 1 mL 1 M magnesium sulfate solution, pH 7.0-7.2, autoclave at 121°C for 20 min, cool to about 60°C and add 1 mL 50% sterile glucose.

[0030] The trace element solutions in the above culture media are specifically: EDTA 50.0 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 2.1 g / L, FeSO4·7H2O 5.0 g / L, CuSO4·5H2O 1.6 g / L, and CoCl2·6H2O 1.6 g / L.

[0031] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0032] Example 1 The Acinetobacter baumannii N-1 provided by the present invention is isolated from the activated sludge of an anoxic tank of a sewage treatment plant in Wuhan, and the process includes the following steps: (1) Enrichment and separation of aerobic denitrifying bacteria.

[0033] The separation method includes the following operations: 1 g sample is weighed from the activated sludge and inoculated into 250 mL of denitrification enrichment medium, and then placed in a constant temperature shaking incubator for 72 h (28 ° C, 180 r / min). 1 mL of the enrichment solution is taken for 10-fold gradient dilution, and 10 -5 -10 -7 100 μL of the dilution solution was evenly spread on the BTB solid culture medium plate, and the plate was placed in a 28°C constant temperature incubator for 48 hours. The plate was observed, and a single clone with a more obvious blue halo on the plate was selected. The strain was streaked and purified and stored in a -80°C ultra-low temperature refrigerator.

[0034] (2) Determination of aerobic denitrification activity.

[0035] The preserved strain was transferred to a culture tube filled with 5 mL of denitrification assay medium, and then placed in a constant temperature shaking incubator for 48 h (28 ° C, 180 r / min), and 2 mL of the bacterial solution was centrifuged at 12000 r / min for 5 min. The supernatant was aspirated for the determination of total nitrogen (TN) concentration and nitrate nitrogen (NO3 - ) concentration, and calculated the nitrate nitrogen removal rate and total nitrogen removal rate of the strain. Among them, the concentration of nitrate nitrogen and total nitrogen was determined by the method in "Water and Wastewater Monitoring and Analysis Methods" (Beijing: China Environmental Science Press, 4th edition, 2002: 258-282).

[0036] The inventors isolated 18 strains with aerobic denitrification ability from the activated sludge from the above sources, and numbered them N-1 to N-18 in sequence. The results of the aerobic denitrification activity of these 18 strains are as follows: Figure 1 As shown. Figure 1It can be seen that N-1 (nitrate removal rate 81%, total nitrogen removal rate 51%), N-5 (nitrate removal rate 67%, total nitrogen removal rate 46%), N-7 (nitrate removal rate 98%, total nitrogen removal rate 47%), N-8 (nitrate removal rate 83%, total nitrogen removal rate 34%) have a relatively efficient denitrification capacity. However, the culture fluids of strains N-5, N-7 and N-8 are turbid and uniform, while the bacteria in the culture fluid of strain N-1 are aggregated flocs, that is, only strain N-1 has the ability to form bacterial flocs.

[0037] (3) Morphological identification of strain N-1.

[0038] The appearance of strain N-1 on R2A solid medium plates and in liquid suspension was observed and recorded. Figure 2 a and Figure 2 As shown in b. From the above results, it can be seen that strain N-1 appears yellow-white, opaque, and the surface of the colony is moist and has a certain stickiness on the solid culture medium plate ( Figure 2 a), Bacterial suspensions are characterized by large and distinct bacterial flocs ( Figure 2 b).

[0039] The size and floc formation characteristics of the strain were further observed using phase contrast microscopy. Figure 2 As shown in c: under the condition of 1000 times magnification, it was found that the bacteria were rod-shaped, with a length of 1.41-1.91μm and a width of 0.75-1.22μm. At the same time, a large number of bacteria could be observed to aggregate into clusters.

[0040] Gram staining was used to identify the strain as Gram-positive bacteria (G + ) or Gram-negative bacteria (G - The identification results showed that the strain was red ( Figure 2 d) The bacteria were identified as Gram-negative bacteria.

[0041] (4) Molecular biological identification.

[0042] The genome of strain N-1 was extracted and used as a template for PCR amplification using universal primers 27F and 1492R. The PCR products were then sent to a biological company for sequencing. The sequencing results were compared for sequence homology using BLAST on the NCBI website and a phylogenetic tree was constructed using MEGA-X software.

[0043] After BLAST comparison of the 16S rDNA sequence obtained after amplification and sequencing (as shown in SEQ ID NO.1), 8 strains were found to have high homology with the 16S rDNA sequence of the strain. In addition, Escherichia coli Escherichia coli (G - ) and Staphylococcus aureusStaphylococcus aureus (G + ) were used to construct a phylogenetic tree using MEGA-X software. The results showed that strain N-1 was closely related to Acinetobacter baumannii ( Acinetobacter baumannii ) on the same branch, the homology similarity is 99.78% ( Figure 3 ).

[0044] Combining the morphological and molecular identification results, strain N-1 was identified as Acinetobacter baumannii and named Acinetobacter baumannii N-1. Acinetobacter sp.N-1.

[0045] Example 2 In order to increase the strain Acinetobacter sp.N-1 denitrification capacity. In this case, we selected three factors, pH, temperature, and C / N, in the denitrification process and used Design Expert to design a response surface experiment to optimize the strain. Acinetobacter Denitrification conditions of sp.N-1.

[0046] Taking the total nitrogen removal rate as the experimental value, a three-factor three-level experimental scheme was set according to the Box-Behnken experimental principle, and the factor level settings are shown in Table 1. The bacterial liquids with different ratios were added to the conical flasks of the same batch filled with 100 mL of denitrification assay medium, placed in a constant temperature shaking incubator and cultured for 48 hours according to the set conditions, and then 2 mL of the sample was taken and centrifuged at 12000 r / min for 5 minutes, and the supernatant was aspirated to determine the total nitrogen concentration of the water sample.

[0047] Table 1 Box-Behnken experimental factors and levels

[0048] strains under various conditions Acinetobacter The total nitrogen removal efficiency of sp.N-1 is shown in Table 2. As can be seen from Table 2, the total nitrogen removal efficiency under different conditions varies greatly (1.64% - 67.67%).

[0049] Table 2 Response surface analysis experimental design and results

[0050] The quadratic fitting regression model was established using Design Expert software, and the regression equation for the total nitrogen removal rate of the strain under different conditions was obtained: Y=60.17+18.02A-16.03B+7.43C-10.31AB+3.80AC-4.95BC-23.08A 2 -13.85B 2 -9.89C 2 ; In the equation, A is pH, B is temperature, and C is C / N. The applicable range of this model is pH: 5-9, temperature: 25-37℃, and C / N: 5-15. Further analysis of variance showed that the R 校正 ²=0.9852, P -value<0.0001, indicating that the model is highly significant; lack of fit term = 0.3284>0.05, indicating that the model regression equation fits well, that is, the model is stable and usable (Table 3). F -value shows that in this model, the influence of the three factors on denitrification efficiency is ranked as follows: pH>temperature>C / N (Table 3).

[0051] Table 3 Analysis of variance of TN removal rate model

[0052] Note: ** indicates extremely significant correlation, * indicates significant correlation.

[0053] Then, the regression equation model was established using Design Expert software to obtain a three-dimensional analysis diagram of the effects of three-factor binary interactions on total nitrogen removal efficiency and the corresponding contour diagram ( Figure 4 ), the results predict that when the culture conditions are pH = 8.339, culture temperature = 25.176℃, C / N = 13.737, the strain Acinetobacter sp.N-1 had the highest total nitrogen removal rate, which could reach 76.89%.

[0054] In order to experimentally verify the above predictions, Acinetobacter sp.N-1's efficient denitrification conditions. In this case, the denitrification experiment was carried out under the predicted optimal conditions, and the total nitrogen removal rate was measured to be 74.36±1.04%, with a deviation of 2.53% between the mean and the predicted value.

[0055] In summary, the present invention successfully isolated a strain that has both aerobic denitrification ability and floc formation characteristics. Acinetobacter sp.N-1 is an ideal strain that can be used in actual aquaculture wastewater purification. Moreover, through response surface analysis, the strain Acinetobacter The total nitrogen removal rate of sp.N-1 increased from 51% to 74.357%, and the denitrification effect increased by 45.79%, providing data support for its efficient application in aquaculture wastewater treatment.

[0056] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. An aerobic denitrifying bacterium with flocculent characteristics, specifically Acinetobacter baumannii N-1 ( Acinetobacter sp.N-1), which is deposited in China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20242568.

2. A microbial agent, characterized in that: The invention comprises the aerobic denitrifying bacteria according to claim 1.

3. Use of the aerobic denitrifying bacteria according to claim 1 or the microbial agent according to claim 2 in wastewater treatment.

4. Use of the aerobic denitrifying bacteria according to claim 1 or the microbial agent according to claim 3 in preparing activated sludge.

5. A wastewater treatment method, characterized in that: Denitrification is carried out using the aerobic denitrifying bacteria described in claim 1.

6. The wastewater treatment method according to claim 5, characterized in that: The pH of the wastewater is 7-9.

7. The wastewater treatment method according to claim 5, characterized in that: The temperature of the wastewater is 25-35°C.

8. The wastewater treatment method according to claim 5, characterized in that: In the wastewater, C / N is 5-15.

9. The wastewater treatment method according to claim 5, characterized in that: Wastewater treatment is carried out using the activated sludge process.

10. The wastewater treatment method according to claim 5, characterized in that: The wastewater is aquaculture wastewater.

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