An efficient oligotrophic aerobic denitrifying Acinetobacter strain and its application

Abstract

The present invention discloses a highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3, with a deposit number of CCTCC NO: M20232588. It can be used for nitrogen removal from river water bodies, and the removal rates of total nitrogen and nitrate nitrogen in river water bodies reach 93.1% and 76.1% respectively within 24 hours. It can effectively remove nitrates in oligotrophic water bodies with a low carbon-nitrogen ratio, and can be further developed into a biological denitrification agent, providing a low-carbon and safe biological treatment method for the treatment of nitrates in surface water bodies such as rivers, lakes and reservoirs.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganisms, and in particular to a high-efficiency oligotrophic aerobic denitrifying Acinetobacter strain and application thereof. Background Art

[0002] Excessive influx of nitrogen in natural water bodies can cause problems such as water hypoxia, eutrophication and algae blooms, destroy the stability of the aquatic ecosystem, and endanger the health of aquatic organisms and humans. Among them, nitrate is the most stable form of nitrogen in water and is difficult to remove. Excessive nitrate concentration in drinking water will increase the risk of diseases such as methemoglobinemia, diabetes, spontaneous abortion, thyroid disease and gastric cancer, and nitrate in water bodies can be converted into nitrite in the human body. Nitrite is extremely toxic. Excessive absorption will cause immediate poisoning reactions and destroy the blood's oxygen delivery function, affecting human health. Therefore, reducing nitrate pollution in surface water such as rivers, lakes and reservoirs, protecting surface water resources and ensuring drinking water safety has become an urgent problem to be solved.

[0003] At present, the common methods for removing nitrates mainly include ion exchange, adsorption, chemical treatment and biological methods, etc. Among them, the denitrification of microorganisms can realize the conversion of nitrates into nitrogen gas, which has the advantages of low cost, high efficiency and environmental friendliness.

[0004] The patent specification with publication number CN110656066A discloses a short-range nitrification and denitrification variant Acinetobacter strain DW-10, but it is used for freshwater aquaculture water treatment in an environment with rich nutrient conditions.

[0005] In reality, some micro-polluted water bodies such as rivers and reservoirs are generally oligotrophic water bodies, and water quality indicators such as nitrates and organic matter exceed the requirements of Grade III water in the "China Surface Water Environmental Quality Standard" (GB3838-2002). Micro-polluted water bodies cannot guarantee the denitrification effect of heterotrophic aerobic denitrifying bacteria due to their low pollution level, poor nutrition for microbial growth, and low C / N ratio. The usual method for this type of water is to add some carbon sources to improve the denitrification efficiency, but this method increases operating costs and may also cause secondary pollution.

[0006] Existing related research has isolated microbial strains from reservoir sediments that can perform aerobic denitrification under oligotrophic conditions. Wen Gang et al. isolated a heterotrophic nitrification-aerobic denitrification Acinetobacter junii ZMF5 from reservoir sediments, which has good denitrification ability under oligotrophic environments (Analysis of the nitrogen removal characteristics and nitrogen / carbon balance of oligotrophic aerobic denitrification strains, Environmental Science, May 2020, Vol. 41, No. 5). The patent specification with the publication number CN113373088A discloses an oligotrophic aerobic denitrifying bacterium agent immobilized with Acidovorax sp. strain YD725 and loofah sponge. Currently, the research in this part mainly focuses on naturally isolated strains, and most of them are heterotrophic nitrification-aerobic denitrification strains, with less research on specific aerobic denitrifying bacteria. In addition, there is also less research on the improvement of such strains. The influencing factors in the actual water environment are complex. By using artificial means to artificially improve the strains, the functions of microbial strains can be better exerted in the actual water environment.

[0007] In summary, the present invention uses a targeted strain improvement method to improve the heterotrophic aerobic denitrifying strain, enabling it to adapt to oligotrophic water bodies and effectively perform denitrifying nitrogen removal under low C / N ratio conditions, which is an effective idea for solving nitrogen pollution in slightly polluted water bodies. Summary of the Invention

[0008] The present invention enriches and screens out dominant and highly efficient oligotrophic aerobic denitrifying strains from the activated sludge of sewage treatment plants, and by means of induced mutation and adaptive evolution screening, focuses on enhancing the denitrification ability of microorganisms, significantly enhancing the ability of microorganisms to remove nitrate nitrogen under oligotrophic conditions, thereby providing a strain with high-efficient aerobic denitrification ability, which has good application scenarios for solving problems such as excessive nitrate in low-carbon source slightly polluted water bodies.

[0009] In the first aspect, the present invention provides a highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3, with the deposit number CCTCC NO: M20232588.

[0010] The highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect is deposited in the China Center for Type Culture Collection (CCTCC), with the deposit date of December 18, 2023, and the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0011] The highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect can survive under oligotrophic conditions (carbon source less than 15 mg / L).

[0012] The highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect can be obtained from the original strain through multiple rounds of mutagenesis and adaptive evolution, and has excellent oligotrophic denitrification ability.

[0013] Exemplarily, the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect can be obtained through the following steps:

[0014] S1: Multiple composite mutagenesis treatment of microbial strains;

[0015] S2: Adaptive evolution under oligotrophic conditions;

[0016] S3: Screening, culturing and effect verification of forward mutant strains.

[0017] In one embodiment, in S1, the multiple composite mutagenesis means that after the strain is treated by ultraviolet mutagenesis and completes one round of adaptive evolution, it is treated with diethyl sulfate again, so as to achieve multiple rounds of composite alternating mutagenesis of ultraviolet and diethyl sulfate.

[0018] In one embodiment, in S2, the adaptive evolution means gradually reducing the content of the culture medium in the next round (reducing by 10% each round) during the process.

[0019] In the second aspect, the present invention provides a highly efficient oligotrophic denitrifying biological agent, and the active ingredient of the agent contains the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect.

[0020] In one embodiment, in the highly efficient oligotrophic denitrifying biological agent described in the second aspect, the viable cell concentration of the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 is 10 10 ~10 12 CFU / mL.

[0021] In the third aspect, the present invention provides the application of the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7-3 described in the first aspect or the highly efficient oligotrophic denitrifying biological agent described in the second aspect in denitrification of river water bodies.

[0022] In one embodiment, in the application described in the third aspect, the river water body is a surface water body of Class III, where the COD, nitrate nitrogen, and ammonia nitrogen concentrations are 0-20 mg / L, 0-4 mg / L, and 0.2-1.0 mg / L in sequence, and the dissolved oxygen concentration is 3-5 mg / L.

[0023] In one embodiment, for the application described in the third aspect, the conditions for denitrifying river water body are as follows: pH = 7 - 8, temperature 25 - 40 °C, C / N ratio 5 - 10, and COD concentration less than 20 mg / L.

[0024] In one embodiment, for the application described in the third aspect, the viable cell concentration of the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7 - 3 described in the first aspect or the highly efficient oligotrophic denitrifying biological agent described in the second aspect, which contains the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7 - 3, is 10 10 ~10 12 CFU / mL.

[0025] Fourth aspect, the present invention provides a method for denitrifying river water body, which comprises adding the highly efficient oligotrophic aerobic denitrifying Acinetobacter sp. strain UD7 - 3 described in the first aspect or the highly efficient oligotrophic denitrifying biological agent described in the second aspect into the river water body.

[0026] In one embodiment, for the method for denitrifying river water body described in the fourth aspect, the river water body is a class III surface water body, where the COD, nitrate nitrogen, and ammonia nitrogen concentrations are 0 - 20 mg / L, 0 - 4 mg / L, and 0.2 - 1.0 mg / L respectively, and the dissolved oxygen concentration is 3 - 5 mg / L.

[0027] In one embodiment, for the method for denitrifying river water body described in the fourth aspect, the conditions for denitrifying river water body are as follows: pH = 7 - 8, temperature 25 - 40 °C, C / N ratio 5 - 10, and COD concentration less than 20 mg / L.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The present invention utilizes the Acinetobacter sp. isolated from a sewage treatment plant. This strain has been acclimated in the sewage treatment plant environment for a long time and has high denitrification ability. The present invention uses a high - efficiency strain breeding method, namely alternating mutagenesis and gradient adaptive evolution, to specifically enhance the survival ability and denitrification ability of this strain in oligotrophic slightly polluted water bodies, and expands the applicable range of the strain.

[0030] 2) Compared with the original strain, the positive mutant strain UD7-3 obtained in this invention has stronger denitrification ability in oligotrophic environment. Its nitrate nitrogen removal rate in the screening medium is 92.6%, which is better than 84.1% of the original strain under the same conditions. In addition, compared with the oligotrophic Acinetobacter ZMF5 screened naturally in the literature (nitrate nitrogen removal rate is 88.02%), UD7-3 has higher nitrate nitrogen removal efficiency under the same conditions. Application experiments prove that the removal rates of nitrate nitrogen and total nitrogen in river water by the enhanced strain UD7-3 obtained in this invention reach 93.1% and 76.1% respectively within 24 hours. Thus, it can be seen that UD7-3 has excellent oligotrophic denitrification ability in nutrient-poor river water, and can be further developed into a biological denitrification agent to provide a low-carbon and safe biological treatment method for the treatment of nitrates in surface waters such as rivers, lakes and reservoirs. Description of the Drawings

[0031] Figure 1 It is a phylogenetic tree diagram of the Acinetobacter strain UD7-3 provided by this invention;

[0032] Figure 2 It is a flow chart of the mutagenesis and adaptive evolution method provided by this invention;

[0033] Figure 3 It is a colony morphology photo of the Acinetobacter strain UD7-3 provided by this invention;

[0034] Figure 4 It is a comparison chart of denitrification efficiency between the original strain and UD7-3;

[0035] Figure 5 It is a result chart of the nitrogen removal rate of the Acinetobacter strain UD7-3 provided by this invention for river water. Detailed Embodiments

[0036] The following further elaborates this invention in combination with the drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate this invention and not to limit the scope of this invention.

[0037] For the operation methods without specific conditions indicated in the following embodiments, they are usually in accordance with conventional conditions or the conditions recommended by the manufacturer.

[0038] In this invention, the conditions of oligotrophic water body refer to the surface water of Class III, with COD lower than 20mg / L and C / N of 5-10.

[0039] Example 1: Obtaining and Identification of the Original Strain

[0040] In this embodiment, the activated sludge in the sewage treatment plant is domesticated, enriched, and screened to obtain oligotrophic aerobic denitrifying strains, which are used for subsequent strain improvement. The bacterial liquid in the enrichment medium is diluted and spread on the solid medium, and single colonies are selected for streaking, and the dominant strains with nitrogen removal effect are identified. The specific experimental steps are as follows:

[0041] 1. Prepare the following media

[0042] (1) Oligotrophic denitrifying enrichment medium (per liter of water): sodium acetate 0.5 g, NaNO3 0.1 g, K2HPO4 0.1 g, CaCl2 0.05 g, MgCl2 0.05 g, trace elements 2 mL, pH 7.0 - 7.5.

[0043] (2) Oligotrophic denitrifying screening medium (per liter of water, or also called selective medium): sodium acetate 0.1 g, NaNO3 0.02 g, K2HPO4 0.02 g, CaCl2 0.01 g, MgCl2 0.01 g, trace elements 2 mL, pH 7.0 - 7.5. 1.5 wt% agar is additionally added to the solid medium.

[0044] (3) Trace element formula (per liter of water): EDTA 10 g, ZnSO4·7H2O 0.4 g, MnCl2·4H2O 1.2 g, FeSO4·7H2O 1.0 g, CuSO4·5H2O, CoCl2·6H2O 0.3 g, Na2MoO4·2H2O 0.2 g, CaCl2 0.1 g, and the pH value is adjusted to 6.5 - 6.8 with KOH.

[0045] 2. Screen oligotrophic denitrifying strains

[0046] (1) Sample collection: The activated sludge is taken from a sewage treatment plant in Dongyang City, Zhejiang Province.

[0047] (2) Enrichment culture and separation: Take 10 g of activated sludge and add it to 100 mL of enrichment medium, and culture it in a shaker at 30 °C and 170 rpm for 72 h.

[0048] (3) Take 10 mL of the enrichment solution for gradient dilution, and take 100 μL of different gradient dilution solutions and spread them on the screening medium, and then invert and culture them in a constant temperature medium at 37 °C. After single colonies grow on the solid plate, select different single colonies for isolation and purification.

[0049] 3. Molecular biological identification of strains

[0050] (1) Genomic DNA was extracted using the Bacterial Genomic DNA Extraction Kit (R403-01) from Nanjing Novoprotein Co., Ltd., and the extraction experiment was carried out according to the steps in the manufacturer's instruction manual. The 16S rDNA fragment was amplified using the bacterial universal primers 27F and 1492R.

[0051] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1)

[0052] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0053] After the product was recovered by gel extraction, it was sent to a sequencing company for sequencing. The obtained sequence was submitted to the NCBI database for blast alignment, and the similarity with Acinetobacter sp. was 99%. An evolutionary tree was drawn based on the aligned sequences, and the evolutionary tree was drawn using MEGAX software as Figure 1 shown. Combining the sequencing results, the original strain was identified as Acinetobacter sp.

[0054] (2) The sequencing result of the 16S rDNA gene of the original strain is shown in SEQ ID NO: 3.

[0055] Example 2: Strain improvement

[0056] In this example, the strain isolated in Example 1 was subjected to multiple rounds of combined mutagenesis with ultraviolet light and diethyl sulfate (DES), and adaptive evolution was carried out by gradually reducing the medium components and C / N ratio, and finally a forward mutant strain that can tolerate extreme oligotrophy and can perform efficient denitrification under low carbon-nitrogen ratio conditions was obtained. The specific steps are as follows:

[0057] 1. Strain mutagenesis and adaptive evolution, the operation process is shown in Figure 2

[0058] Take 10 mL of the original strain (Acinetobacter sp.) of aerobic denitrifying bacteria isolated from the sewage treatment plant (OD 600(= 1.0) was placed in a 90 mm petri dish on a magnetic stirrer and irradiated with ultraviolet light (ultraviolet irradiation power: 15 W, irradiation distance: 20 cm, irradiation time: 45 - 60 s). After the treatment, the ultraviolet lamp was turned off, 100 μL of the bacterial solution was aspirated and added to the oligotrophic screening medium (90 vol% medium + 10 vol% water), and cultured in the dark at 30 °C on a shaker at 170 rpm. After 24 h, subculture was carried out, and at least 5 subculture processes were repeated to complete one round of mutagenesis culture. The bacterial solution obtained from one round of mutagenesis was used as the starting bacterial solution for the next experiment. For the second round, it was treated with DES. After aspirating 1 mL of the bacterial solution, 0.4 wt% of DES was added. After 20 min of treatment, 0.1 mL of sodium thiosulfate with a concentration of 25 wt% was added to terminate the reaction. 100 μL of the treated bacterial solution was aspirated and transferred to the screening medium (80% vol medium + 20% vol water), and the culture and transfer were repeated 5 times. The above experimental process was repeated (the volume ratio of the acclimation medium was reduced by 10% in each round), and alternating mutagenesis with ultraviolet light and DES was carried out (to prevent microorganisms from enhancing their tolerance to mutagenic factors). Finally, it was reduced to 10 vol% medium + 90 vol% water, and the strain could not grow.

[0059] The conditions of the above ultraviolet and DES treatments are determined according to the tolerance of different microorganisms (prokaryotic or eukaryotic) to ultraviolet light and DES. Preliminary experiments need to be carried out to explore suitable mutagenesis conditions to ensure that the lethality after treatment remains at about 70% - 80%.

[0060] 2. Screening of positive mutant strains

[0061] The bacterial solution obtained after the above multiple rounds of mutagenesis and adaptive evolution was subjected to gradient dilution and spread plating on solid plates. After colonies grew on the solid plates, the colony morphology was observed every day. Colonies with good growth were selected and cultured in the screening medium. Colonies with good growth were continuously selected and transferred to the screening medium, and cultured at 30 °C on a shaker at 170 rpm. The nitrate concentration was detected every 24 h to screen out strains with better denitrification effects. After screening, a dominant aerobic denitrifying positive mutant strain under oligotrophic conditions was obtained. As Figure 4 shown, its denitrification efficiency was better than that of the original strain. After 24 h of treatment, the nitrate nitrogen decreased from 3.09 mg / L to 0.23 mg / L (for the original strain it was 0.49 mg / L), and the degradation rate was 92.6%, which was better than 84.1% of the original strain. This strain was named UD7 - 3.

[0062] The isolated UD7 - 3 strain was streaked on an LB solid medium (agar), and after culturing in an incubator at 30 °C for 48 h, its colony size, color, edge, smoothness, transparency and other characteristics were observed. As Figure 3 shown, this strain formed round, irregular - edged, smooth, moist, and opaque yellow colonies on the LB solid medium.

[0063] This strain was deposited at the China Center for Type Culture Collection on December 18, 2023, with the deposit number CCTCC NO: M20232588.

[0064] Example 3: Application of Strain UD7-3 in Denitrification of Natural River Water

[0065] In this example, natural river water was used. The water quality of this river water was as follows: total nitrogen 2.64 mg / L, ammonia nitrogen 0.86 mg / L, nitrate nitrogen 1.74 mg / L, COD 15.4 mg / L, C / N ratio about 5.8, and water body pH value about 7.2. The fermentation broth of UD7-3 above (bacterial amount was 10 12 CFU / mL) was inoculated into the river water at an inoculation amount of 2%, and after culturing at 25 °C with a shaker rotation speed of 170 rpm for 24 h, the changes in the contents of total nitrogen and nitrate nitrogen in the water body were measured.

[0066] See Figure 5 , after 24 h of culture, the concentration of nitrate nitrogen decreased from the initial concentration of 1.74 mg / L to 0.12 mg / L at 24 h, the nitrate removal rate reached 93.1%, and the total nitrogen removal rate reached 76.1%, showing excellent denitrification and nitrogen removal effects in oligotrophic water bodies. In addition, it should be noted that the original strain had no ammonia nitrogen degradation ability, and from the above nitrate and total nitrogen removal rates, it can be seen that the forward mutant strain UD7-3 obtained in the present invention not only has a more excellent denitrification and nitrogen removal ability in oligotrophic water bodies than the original strain, but also shows the ability to remove other forms of nitrogen such as ammonia nitrogen.

[0067] The detection methods in the above effect verification: the detection method for nitrate nitrogen was to determine nitrate nitrogen by ultraviolet spectrophotometry (HJ / T346-2007); the detection method for total nitrogen was alkaline potassium persulfate digestion ultraviolet spectrophotometry (HJ636-2012).

[0068] In addition, it should be understood that after reading the above description content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A highly efficient oligotrophic aerobic denitrifying Acinetobacter ( Acinetobacter sp.) strain UD7-3, characterized in that The deposit number is CCTCC NO: M20232588.

2. A highly efficient oligotrophic denitrification biological agent, characterized in that: The active ingredients of the bacterial agent include the highly efficient oligotrophic aerobic denitrifying Acinetobacter described in claim 1 ( Acinetobacter sp.) strain UD7-3.

3. The high-efficiency oligotrophic denitrification biological agent according to claim 2, characterized in that: The highly efficient oligotrophic aerobic denitrifying Acinetobacter ( Acinetobacter sp.) strain UD7-3 had a live bacterial concentration of 10 10 ~10 12 CFU / mL.

4. The highly efficient oligotrophic aerobic denitrifying Acinetobacter according to claim 1 ( Acinetobacter sp.) strain UD7-3 or the high-efficiency oligotrophic denitrification biological agent according to claim 2 or 3 in denitrification of river water, characterized in that: The conditions for denitrification of river water include: a C / N ratio of 5 to 10 and a COD lower than 20 mg / L.

5. The use according to claim 4, characterized in that: The river water body is a Class III surface water body, with a nitrate nitrogen concentration of 1.74~4 mg / L, an ammonia nitrogen concentration of 0.2~1.0 mg / L, and a dissolved oxygen concentration of 3~5 mg / L.

6. The use according to claim 4, characterized in that: The conditions for denitrification of river water also include: pH=7~8, temperature 25~40℃.

7. A method for denitrifying river water, characterized in that: Add the highly efficient oligotrophic aerobic denitrifying Acinetobacter ( Acinetobacter sp.) strain UD7-3 or the highly efficient oligotrophic denitrification biological agent according to claim 2 or 3; The conditions for denitrification of river water include: a C / N ratio of 5 to 10 and a COD lower than 20 mg / L.

8. The method for denitrifying river water according to claim 7, characterized in that: The river water body is Class III surface water, with a nitrate nitrogen concentration of 1.74~4 mg / L, an ammonia nitrogen concentration of 0.2~1.0 mg / L, and a dissolved oxygen concentration of 3~5 mg / L.

9. The method for denitrifying river water according to claim 7, characterized in that: The conditions for denitrification of river water also include: pH=7~8, temperature 25~40°C.

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