Aerobic denitrification actinomycete strain and use thereof

Abstract

Provided are an aerobic denitrification actinomycete strain and a use thereof. The strain is named Streptomyces venezuelae D18, and the aerobic denitrification actinomycete strain can be used for water body remediation. The aerobic denitrification actinomycete strain has a dissolved organic carbon removal rate of 93.34%, a nitrate removal rate of 99.79%, and a total nitrogen removal rate of 98.44%, and the strain has good denitrification performance, and provides a new insight for developing diversified efficient aerobic denitrification biological resources. When the strain is used for water body remediation, the total nitrogen removal rate can reach 89.16%, and the permanganate removal rate can reach 91.44%. The strain can efficiently realize remediation of lightly polluted water bodies.

Description

Aerobic denitrifying actinomycetes and application thereof Technical Field

[0001] The present invention belongs to the technical field of water pollution control, relates to microbial control of water pollution, and specifically relates to an aerobic denitrifying actinomycete and an application thereof. Background Art

[0002] In natural aquatic ecosystems, water bodies are often slightly polluted, with pollutant concentrations (such as nitrates and organic matter) below 10 mg / L. Physical and chemical methods are often not cost-effective for removing small amounts of pollutants from these water bodies. Biological denitrification is a more economical option. Biological denitrification involves aerobic denitrification, which has a strong tolerance to dissolved oxygen, moderate costs, high efficiency, and clean, environmentally friendly byproducts, making it a hot topic in denitrification research.

[0003] Actinomycetes are a type of microorganism with aerobic denitrification capabilities that can be used to repair farmland water bodies and slightly polluted water bodies. Streptomyces is a large genus of Actinobacteria that has great potential in slightly polluted water bodies. Screening and obtaining Streptomyces with efficient aerobic denitrification capabilities will help develop a variety of efficient aerobic denitrification biological resources and is of great significance for further improving the denitrification efficiency and economy of slightly polluted water bodies.

[0004] Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an aerobic denitrifying actinomycete and its application to solve the technical problem that the efficiency of biological denitrification treatment of slightly polluted water bodies in the existing technology needs to be further improved.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An aerobic denitrifying actinomycete, the strain is named Streptomyces venezuelae D18, and is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M20231417. The deposit date is August 10, 2023, and the deposit address is Wuhan.

[0008] The present invention also has the following technical features:

[0009] The pH value of the culture medium of the aerobic denitrifying actinomycetes is 7, the only carbon source in the culture medium is soluble starch, the only nitrogen source is nitrate, and the carbon-nitrogen mass ratio (C / N) of the soluble starch and nitrate is 10.

[0010] The aerobic denitrifying actinomycetes were cultured at a temperature of 30° C. and a rotation speed of 150 rpm.

[0011] The present invention also protects the use of the aerobic denitrifying actinomycetes described above for water body remediation.

[0012] The application method includes: inoculating aerobic denitrifying actinomycetes into raw water, continuously oxygenating the raw water, and maintaining the dissolved oxygen concentration in the raw water at 5 to 15 mg / L.

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] The aerobic denitrifying actinomycetes of the present invention can achieve a dissolved organic carbon removal rate of 93.34%, a nitrate removal rate of 99.79%, and a total nitrogen removal rate of 98.44%. This strain has excellent denitrification performance and provides new insights into the development of diverse and efficient aerobic denitrifying biological resources. When used for water remediation, this strain can achieve a total nitrogen removal rate of 89.16% and a permanganate removal rate of 91.44%, effectively remediating slightly polluted water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a phylogenetic tree of aerobic denitrifying actinomycetes (Streptomyces venezuelae D18).

[0016] Figure 2 shows the growth curve and total dissolved organic carbon degradation curve of aerobic denitrifying actinomycetes using nitrate as nitrogen source.

[0017] Figure 3 shows the aerobic denitrification performance of aerobic denitrifying actinomycetes using nitrate as the only nitrogen source.

[0018] Figure 4 is a graph showing the aerobic denitrification performance of aerobic denitrifying actinomycetes under different conditions when nitrate is the sole nitrogen source; in Figure 4: a represents different carbon sources (the carbon sources are soluble starch, glucose, sucrose, sodium succinate, sodium acetate and sodium citrate); b represents different carbon-nitrogen ratios (C / N is 1, 3, 5, 7 and 9); c represents different pH values ​​(pH values ​​are 4, 5, 6, 7, 8, 9); d represents different rotational speeds (rotational speeds are 0 rpm, 20 rpm, 40 rpm, 60 rpm, 80 rpm, 100 rpm, 120 rpm).

[0019] FIG5 is a line graph showing the cell growth and pollutant removal in the raw water of the landscape water body of Example 2; FIG5 a is a cell density curve, a permanganate removal curve and a histogram of the total number of cells, and b is a histogram of total nitrogen (TN), ammonia nitrogen (NH4 + -N), nitrate (NO3 ‐ -N) and nitrite (NO2‐ -N)Removal curve graph.

[0020] FIG6 is a line graph showing the cell growth and pollutant removal in the raw water of the reservoir in Example 3; FIG6 a is a cell density curve, a permanganate removal curve, and a histogram of the total number of cells; and b is a histogram showing the total nitrogen (TN), ammonia nitrogen (NH4 + -N), nitrate (NO3 ‐ -N) and nitrite (NO2 ‐ -N)Removal curve graph.

[0021] The specific contents of the present invention are further explained in detail below with reference to the embodiments. DETAILED DESCRIPTION

[0022] It should be noted that all reagents and culture media used in the present invention, unless otherwise specified, are reagents and culture media known in the art, for example:

[0023] The actinomycete liquid medium and solid medium for actinomycetes were Gao's No. 1 liquid medium and Gao's No. 1 solid medium, both known in the art, with a formulation of 1 g / L KNO₃, 0.5 g / L NaCl, 0.01 g / L FeSO₄, 0.5 g / L KH₂PO₄, 0.5 g / L MgSO₄, and 20 g / L soluble starch, with a pH of 7.1-7.3. The actinomycete solid medium was prepared by adding 20 g / L of agar powder to the aforementioned formulation.

[0024] The denitrification liquid culture medium adopts a conventional denitrification liquid culture medium known in the prior art, and its formula and preparation method are as follows: 0.108 g / L of KNO3, 1.5 g / L of KH2PO4, 0.413 g / L of glucose, 0.1 g / L of MgSO4·7H2O, 5.0 g / L of Na2HPO4·12H2O, and 2 mL of trace element mother solution; the above components are added to ultrapure water and the volume is adjusted to 1 L, stirred until completely dissolved, and then the pH value is adjusted to 7.0-7.2. The mixture is sterilized at 121°C for 30 minutes before use.

[0025] The formula and preparation method of the above trace element mother solution are as follows: 4.4 mg ZnSO4, 100 mg EDTA, 10.2 mg MnCl2·4H2O, 11 mg CaCl2, 10 mg FeSO4·7H2O, 3.2 mg CuSO4·5H2O, 2.2 mg (NH4)6Mo7O 24·4H2O, 3.2mg of CoCl2·6H2O; add the above components to ultrapure water and make up to 1L, stir until completely dissolved, then adjust the pH value to 7.0-7.2, and sterilize at 121℃ for 30 minutes before use.

[0026] Specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made on the basis of the technical solution of this application fall within the protection scope of the present invention.

[0027] Example 1:

[0028] This example provides an aerobic denitrifying actinomycete, which is named Streptomyces venezuelae D18, the Chinese genus name is Venezuelan Streptomyces, and is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M20231417, a deposit date of August 10, 2023, and a deposit address of Wuhan.

[0029] In this embodiment, the screening and identification method of aerobic denitrifying actinomycetes specifically includes the following steps:

[0030] Step 1: Enrichment of aerobic denitrifying actinomycetes:

[0031] A 100 mL sample of overlying water from a landscape water body was filtered through a polycarbonate membrane (0.22 μm). The membrane was then immediately placed on a solid culture medium containing actinomycetes containing K₂Cr₂Oₐ (final concentration: 80 ppm). The samples were then cultured using the pure culture method at 30±2°C for 6–9 days. All samples were cultured in triplicate (n=3). Actinomycetes can pass through the polycarbonate membrane and grow on the solid culture medium, while the addition of K₂Cr₂Oₐ inhibits the competitive growth of bacteria and fungi on the solid culture medium.

[0032] Step 2: Initial screening of aerobic denitrifying actinomycetes:

[0033] Take the actinomycete solid culture medium obtained after enrichment in step 1 and streak dry single colonies of the same color, shape, and size onto the solid culture medium. Incubate the culture medium at 30±2°C for 7-12 days using the pure culture method. All samples were cultured in triplicate (n=3). After 5-6 purification cycles, multiple pure colonies of the initially screened actinomycetes were obtained.

[0034] Step 3: Rescreening of aerobic denitrifying actinomycetes:

[0035] Inoculate the multiple pure bacterial colonies obtained in step 2 into an actinomycete liquid culture medium and culture at 135 rpm and 30°C for 5 days to prepare a seed solution. Add 6% of the seed solution by volume to a denitrification assay medium and culture at 135 rpm and 30°C with shaking. After 72 hours, select strains with a total nitrogen removal rate of 95% or higher as the test strains.

[0036] Step 4: strain identification:

[0037] The genomic DNA of the test strain obtained in step 3 was used as a template for PCR identification of the test strain. The genomic DNA of the test strain was extracted using an Omega DNA kit (Biotechnology, USA). The upstream primer used for PCR identification was 27F, whose sequence was 5′-AGTTTGATCMTGGCTCAG-3′; the downstream primer used for PCR identification was 1492R, whose sequence was 5′-CGGCCGCGGCTGCTGGCACGT-3′.

[0038] The PCR reaction system (25 μL) consisted of 20-50 ng / μL genomic DNA template, 12.5 μL PCR premix, 1 μL primer 27F (10 μM), 1 μL primer 1492R (10 μM), and 9.5 μL ddH2O. The PCR program was as follows: 95°C for 5 min; 94°C for 30 s; 57°C for 30 s; 72°C for 90 s; 30 cycles of amplification followed by 72°C for 10 min. After PCR, the 16S rDNA fragment was obtained, and its 16S rDNA nucleotide sequence was determined by sequencing as follows:

[0039] The alignment results of the above 16S rDNA nucleotide sequence are shown in Figure 1. As shown in Figure 1, the 16S rDNA nucleotide sequence is more than 99% similar to Streptomyces sp. ATCC 15068, Streptomyces sp. RG8H, and Streptomyces sp. A1013Y, belonging to the genus Streptomyces sp., and thus the test strain was determined to be Streptomyces venezuelae (Venezuela Streptomyces), and was named Streptomyces venezuelae D18.

[0040] In this example, the aerobic denitrification ability of aerobic denitrifying actinomycetes (Streptomyces venezuelae D18) was tested as follows:

[0041] The aerobic denitrifying actinomycete seed solution was inoculated into the denitrification liquid medium at a volume ratio of 1%, and cultured at 30°C and 135 rpm for 48 hours. During this period, samples were taken every 3 hours to measure the optical density (OD 600 ), total nitrogen (TN), ammonia nitrogen (NH4 + -N), nitrate (NO3 ‐ -N), nitrite (NO2 ‐ -N) and dissolved organic carbon (DOC) concentrations, and the measurement results are shown in Figures 2 to 4

[0042] As shown in Figure 2, the strain was in the lag phase from 0 to 6 hours, the logarithmic phase from 6 to 39 hours, and the stationary phase after 39 hours of culture. The dissolved organic carbon concentration of the strain gradually decreased during the culture period. After 48 hours of culture, strain D18 was cultured in a medium with an initial dissolved organic carbon concentration of 150 mg / L, and the dissolved organic carbon removal rate was 93.34%. In addition, the Pearson correlation index confirmed that the dissolved organic carbon concentration was significantly negatively correlated with cell growth (r = -0.983, P < 0.001, n = 17), indicating that the carbon source can provide the energy and electron donor required for growth and metabolism of the strain.

[0043] As shown in Figure 3, after 48 hours of culture, the nitrate removal rate was 99.79% and the total nitrogen removal rate was 98.44%. There was no obvious accumulation of nitrite and ammonia nitrogen during the process. This result shows that the strain has obvious aerobic denitrification ability.

[0044] As shown in Figure 4, the strain has strong adaptability to the environment. The optimal culture conditions of the strain are: soluble starch as the carbon source, nitrate as the nitrogen source, a carbon-nitrogen ratio of 10 (the carbon-nitrogen ratio refers to the mass ratio of the C element in the carbon source to the nitrogen element in the nitrogen source), a temperature of 30°C, a rotation speed of 150 rpm, and a neutral environment with a pH value of 7.

[0045] Example 2:

[0046] This example describes the application of the aerobic denitrifying actinomycetes of Example 1 in water body remediation. The specific process of this application is: a seed solution of aerobic denitrifying actinomycetes is added to an aerobic reactor containing raw water from a landscape water body at a volume ratio of 2.5%, and an oxygen pump is used to provide continuous aeration for the aerobic reactor so that the dissolved oxygen concentration in the raw water is maintained at 5 to 15 mg / L.

[0047] In this embodiment, the raw water sample of the landscape water body was taken from the Xi'an City Sports Park (34°20′41″N, 108°56′25″E).

[0048] In this embodiment, the permanganate index (COD Mn ), ammonia nitrogen (NH4 + -N), nitrate (NO3 ‐ -N), nitrite (NO2 ‐ ‐N) and total nitrogen (TN) removal, as well as the total cell count and OD 600 The values ​​were tested, and the test results are shown in Figure 5.

[0049] As shown in Figure 5, in the raw water of the landscape water body, strain D18 reduced the total nitrogen concentration from 7.12 mg / L to 1.09 mg / L, and the total nitrogen removal rate was 84.27%. 600 In a complex raw water environment, the strain D18 showed an increasing trend. After the eighth day of reaction, the total cell content in the reactor increased by 8.89 times. In addition, the strain D18 reduced the permanganate concentration from 6.94 mg / L to 0.56 mg / L, with a permanganate removal rate of 91.44%.

[0050] Example 3:

[0051] This example describes the use of the aerobic denitrifying actinomycetes of Example 1 for water remediation. The specific process of this application is as follows: a seed solution of aerobic denitrifying actinomycetes is added to an aerobic reactor containing raw water from a water source reservoir at a volume ratio of 2.5%, and an oxygen pump is used to provide continuous aeration for the aerobic reactor so that the dissolved oxygen concentration in the raw water is maintained at 5 to 15 mg / L.

[0052] In this embodiment, the raw water sample of the water source reservoir was taken from the Xili Reservoir in Shenzhen City (113°57′6″E, 22°35′37″N).

[0053] In this embodiment, the permanganate index (COD Mn ), ammonia nitrogen (NH4 + -N), nitrate (NO3 ‐ -N), nitrite (NO2 ‐ ‐N) and total nitrogen (TN) removal, as well as the total cell count and OD 600 The values ​​were tested, and the test results are shown in Figure 6.

[0054] As shown in Figure 6, in the raw water of the source reservoir, the total nitrogen concentration decreased from 3.18 mg / L to 0.75 mg / L before and after adding the seed solution, and the total nitrogen removal rate was 89.16%. The permanganate concentration decreased from 3.35 mg / L to 0.59 mg / L, and the permanganate removal rate was 82.39%. In addition, OD 600 In the complex raw water environment, it showed an increasing trend, and the OD 600The value reached 0.087, and the total number of cells in the reactor increased by 4.7 times.

[0055] Based on the results of Examples 1 to 3, it can be seen that Streptomyces venezuelae D18 has both excellent aerobic denitrification performance and the effect of dissolved organic carbon concentration. Streptomyces venezuelae D188 has strong adaptability in raw water environments and can utilize the complex carbon sources in landscape water bodies and water source reservoirs to maintain continuous cell growth. This strain can effectively reduce the content of ammonia nitrogen, nitrate, nitrite and COD in slightly polluted water, which has broad application prospects for the development of denitrifying bacteria agents or sewage treatment agents. Therefore, strain D18 has great application potential in the in-situ treatment of slightly polluted lake and reservoir water bodies.

Claims

1. An aerobic denitrifying actinomycete, characterized in that: It was named Streptomyces venezuelae D18 and deposited in China Center for Type Culture Collection (CCTCC) with the accession number CCTCC NO:M20231417.

2. The aerobic denitrifying actinomycetes according to claim 1, characterized in that The pH value of the culture medium of the aerobic denitrifying actinomycetes is 7, the only carbon source in the culture medium is soluble starch, the only nitrogen source is nitrate, and the carbon-nitrogen ratio is 10.

3. The aerobic denitrifying actinomycetes according to claim 1, characterized in that The culture temperature of the aerobic denitrifying actinomycetes was 30° C. and the rotation speed was 150 rpm.

4. Use of the aerobic denitrifying actinomycetes according to any one of claims 1 to 3 for water body remediation.

5. The use according to claim 4, characterized in that The application method includes: inoculating aerobic denitrifying actinomycetes into raw water, continuously oxygenating the raw water, and maintaining the dissolved oxygen concentration in the raw water at 5 to 15 mg / L.

Images