High-ammonia-nitrogen-tolerant heterotrophic nitrifying bacteria and application thereof

Abstract

The application discloses a high-ammonia-nitrogen-tolerant heterotrophic nitrifying bacterium and application thereof. The bacterium is named as heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13, and the preservation number is GDMCC No: 60528. The heterotrophic nitrifying bacterium ZFS1-13 grows well in simulated high-ammonia-nitrogen wastewater with an initial ammonia-nitrogen concentration of 1500 mg / L, and still maintains partial ammonia-nitrogen removal capacity. After being cultured in a denitrification culture medium with an initial ammonia-nitrogen concentration of 150 mg / L at 180 rpm and 28 DEG C for 48 h, the ammonia-nitrogen removal rate is greater than 60%. Therefore, the heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13 of the application can be used for treating ammonia-nitrogen wastewater, can tolerate high-concentration ammonia-nitrogen, and has important significance for the development of denitrification technology and process.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and environmental science, specifically to a strain of heterotrophic nitrifying bacteria tolerant to high ammonia nitrogen and its applications. Background Technology

[0002] Currently, water pollution in my country is becoming increasingly serious. There are many causes of water environment deterioration, among which the direct discharge of high-ammonia nitrogen wastewater from landfill leachate, aquaculture wastewater, and coking wastewater without effective treatment is a significant contributor. The input of high-ammonia nitrogen wastewater leads to eutrophication of lakes, the death of aquatic organisms, and disrupts the normal ecosystem balance, causing serious harm to the ecological environment.

[0003] Currently, biological denitrification technology has become a hot research topic in the field of water treatment, possessing advantages such as low cost, high efficiency, ease of operation, and no secondary pollution, and has great development potential. However, current biological denitrification technology faces significant challenges in treating high ammonia nitrogen wastewater. The main reason is that high ammonia nitrogen wastewater has a strong direct toxic effect on microorganisms, making it difficult for microorganisms to colonize and grow in the high ammonia nitrogen wastewater environment, thus causing them to lose their denitrification capacity.

[0004] Traditionally, nitrification is considered to be a nitrification process carried out by autotrophic bacteria under aerobic conditions (NH4+). + →NH2OH→NO2 - →NO3 - With the discovery of heterotrophic nitrifying bacteria, it has been recognized that nitrification can also occur in heterotrophic nitrifying bacteria. In recent years, many studies have shown that, compared to autotrophic nitrifying bacteria, heterotrophic nitrifying bacteria have faster growth rates, higher cell yields, require lower dissolved oxygen concentrations, and are more acid-resistant. Therefore, isolating novel and efficient heterotrophic nitrifying bacteria from nature and applying them to the treatment of wastewater with high ammonia nitrogen concentrations is of great significance to the development of denitrification technologies and processes.

[0005] Currently reported heterotrophic nitrifying bacteria belong to genera such as *Arthrobacter*, *Flavobacterium*, *Achromobacter*, *Zobellella*, *Serratia*, *Pseudomonas*, *Alcaligenes*, *Paracoccus*, *Bacillus*, *Enterobacter*, and *Sphingomonas*. No heterotrophic nitrifying bacteria have yet been found in the genus *Sphingomonas*. Summary of the Invention

[0006] The purpose of this invention is to provide a heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13 and a microbial agent containing said heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13, providing a good biological material for denitrification of water bodies.

[0007] Another object of the present invention is to provide the application of the heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 and the corresponding microbial agents in the removal of ammonia nitrogen, particularly in the removal of ammonia nitrogen from water bodies.

[0008] To achieve the above objectives, this invention discloses a high-ammonia nitrogen-tolerant heterotrophic nitrifying bacterium, *Sphingobacterium sp.*, isolated and screened from wastewater in a natural aeration tank for pig manure. The strain is named ZFS1-13 and belongs to the phylum Bacteroidetes and family Sphingobacteriaceae. It is named *Sphingobacterium sp.* ZFS1-13. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 60528 and deposit date of December 20, 2018.

[0009] The heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 of this invention was isolated by the inventors in February 2018 from a wastewater sample from a natural aeration tank containing pig manure after 8 months of domestication and enrichment. The physiological and biochemical characteristics of this strain are: Gram-negative bacteria, rod-shaped cells, non-spore-forming, non-flagellated, with a thick, sticky outer layer covering the cell body, and a size of 0.5–1.0 × 0.8–2.0 μm (see...). Figure 2 The colonies are light yellow, smooth, round, convex in the center, and with neat edges (see...). Figure 1 ).

[0010] The nucleotide sequence of the 16S rRNA of the heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13 of the present invention is shown in SEQ ID NO:1. In the NCBI / GenBank database, its 16S rRNA gene sequence shows the highest similarity (94%) to Sphingobacterium suaedae strain T47 among currently isolated bacteria.

[0011] The heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 of the present invention grows well in simulated wastewater with a high ammonia nitrogen concentration of 1500 mg / L. After 48 h of cultivation, the OD600 value reaches 0.85 and the ammonia nitrogen removal rate is 12.8%.

[0012] The heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 of this invention, after being cultured at 180 rpm and 28℃ for 48 h in a denitrification medium with ammonia nitrogen as the sole nitrogen source, achieved a nitrate nitrogen removal rate of more than 60%. Therefore, it can be concluded that the heterotrophic nitrifying bacteria ZFS1-13 can be used to treat ammonia nitrogen wastewater and remove ammonia nitrogen.

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

[0014] This invention discovers a novel heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13. This strain grows well in simulated high ammonia nitrogen wastewater with an initial ammonia nitrogen concentration of 1500 mg / L, while still retaining some ammonia nitrogen removal capacity. After being cultured for 48 hours at 180 rpm and 28°C in a denitrification medium with an initial ammonia nitrogen concentration of 150 mg / L, the ammonia nitrogen removal rate is greater than 60%. Therefore, the heterotrophic nitrifying bacterium (Sphingobacterium sp.) ZFS1-13 of this invention can be used to treat ammonia nitrogen wastewater, which is of great significance to the development of denitrification technology and processes.

[0015] The heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 of the present invention was deposited on December 20, 2018 at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 60528. Attached Figure Description

[0016] Figure 1 The colony morphology of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 on solid plates.

[0017] Figure 2 Photograph (A) and transmission electron micrograph (B) of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 after crystal violet staining under an optical microscope (100×).

[0018] Figure 3Phylogenetic trees of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 and representative strains of all genera within the same family; the NJ method was used to construct the trees, and only bootstrap coefficients >70% (repeated 1000 times) are shown in the figures.

[0019] Figure 4 Phylogenetic trees of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 and related strains are shown; the NJ method was used to construct the trees, and only bootstrap coefficients >70% (repeated 1000 times) are shown in the figures.

[0020] Figure 5 This is a schematic diagram showing the growth trend and denitrification curve of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 under different initial ammonia nitrogen concentrations.

[0021] Figure 6 This is a schematic diagram of the growth trend and denitrification curve of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 at an initial ammonia nitrogen concentration of 150 mg / L. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The parameters, proportions, etc. of the examples can be selected according to local conditions without substantially affecting the results. Unless otherwise specified, the methods described in the examples are all conventional methods, and the reagents used are all conventional reagents or reagents prepared in a conventional manner.

[0023] Example 1: Isolation of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13

[0024] Wastewater samples from the natural aeration pond of pig manure were collected from the primary natural aeration pond of the pig farm of Jinruifeng Ecological Agriculture Co., Ltd. in Shanwei City, Guangdong Province. 100 mL of pig manure was added to each bottle, along with 10 g of sterilized pig manure. The mixture was incubated at 30℃ and 180 rpm, with water replenished weekly to 100 mL; 10 g of sterilized pig manure was added every two weeks. Acclimation culture began on February 21, 2018, and isolation was achieved on October 26, 2018, with enrichment and acclimation culture lasting approximately 8 months. After acclimation culture, the pig manure water samples were used, and the diluted suspension was spread onto Reasoner's 2A agar (R2A, Reasoner's 2A) plates using the standard dilution plating method. The plates were incubated at 30℃ for 3 days. Colonies were then picked from the plates and repeatedly streaked for purification. The colonies of the obtained strain ZFS1-13 are shown below. Figure 1 and Figure 2As shown, the colony is a light yellow, smooth, round shape with a convex center and neat edges. Figure 1 ), a Gram-negative bacterium, with rod-shaped cells, no spores, no flagella, and a thick, sticky outer layer covering the cell body, measuring 0.5–1.0 × 0.8–2.0 μm ( Figure 2 A), Transmission electron microscopy image as shown Figure 2 As shown in B.

[0025] The purified strain ZFS1-13 was preserved in glycerol tubes (-80℃) and lyophilized tubes (4℃). Experiments showed that strain ZFS1-13 is generally cultured using Nutrient Agar or Reasoner's 2A agar at a temperature of 28–30℃.

[0026] Example 2: Identification of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13

[0027] Genomic DNA was extracted from strain ZFS1-13 using the CTAB method. PCR products were amplified using universal primers 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGACTTAACCCCAATCGC-3') and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (Guangzhou Branch) for sequencing. The obtained 16S rRNA sequence was 1401 bp in length, and the nucleotide sequence is shown in SEQ ID NO:1.

[0028] The 16S rRNA gene sequences of strains ZFS1-13 and representative strains of all genera within the same family were selected. Phylogenetic trees were constructed using MEGA 6.0, the Kimura 2-parameter model, and the NJ algorithm (bootstrap repeated 1000 times). The resulting phylogenetic trees are shown below. Figure 3 Then, strain ZFS1-13 and similar strains were selected and a phylogenetic tree was constructed again using the above method. The final phylogenetic tree results are shown in [reference needed]. Figure 4 .

[0029] In the EzBioCloud database, the 16S rRNA gene sequence of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 (i.e., strain ZFS1-13) showed the highest similarity (94.1%) to Sphingobacterium suaedae strain T47T, followed by Sphingobacterium soli YIM X0211T (93.02%), Sphingobacterium composti 2DSM 18850T (92.92%), Sphingobacterium cellulitidis R-53603T (92.75%), and Sphingobacterium tabacisoli h337T (92.75%). Based on the above results, the strain ZFS1-13 isolated in Example 1 of this invention was preliminarily identified as a new species of Sphingobacterium, tentatively named: heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13.

[0030] Example 3: Assessment of the ammonia nitrogen tolerance of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13

[0031] (1) Solution preparation

[0032] The method for preparing the composite carbon source mixture is as follows: Take 6.9g of D-glucose, 6.9g of D-fructose, 6.9g of D-lactose, 9.5g of sodium acetate, 7.0g of mannitol, 4.8g of sodium benzoate, 4.6g of salicylic acid, 6.4mL of 90% v / v lactic acid, 7.0mL of anhydrous ethanol, and 6.3mL of glycerol, dissolve them in 500mL of water, adjust the pH to 7.4, and filter sterilize using a 0.22μm filter membrane.

[0033] The preparation method for the compound vitamin solution is as follows: Take 10mg of biotin / VH, 100mg of folic acid / VB9, 100mg of pyridoxine / VB6, 200mg of thiamine / VB1, 200mg of niacin, 100mg of calcium pantothenate, and VB... 12 Add 2mg of riboflavin / VB2, 20mg of lipoic acid, and 400mg of EDTA-Na to 1L of water, adjust the pH to 7.5, and filter with a 0.22μm filter membrane for sterilization.

[0034] The method for preparing the trace element mixture is as follows: Take 0.1g of ZnSO4·7H2O, 0.4g of MnCl2·4H2O, 1.24g of H3BO3, 0.5g of CoCl2·2H2O, 0.5g of CuCl2·2H2O, 0.02g of NiSO4·6H2O, 0.4g of NaMoO4·2H2O, 0.2g of KI, 5.0g of CaCl2, 1.1g of FeSO4·7H2O, and 10.0g of EDTA-Na, dissolve them in 1000mL of H2O, adjust the pH to 7.4, and filter using a 0.22μm filter membrane.

[0035] The method for preparing simulated wastewater with different ammonia nitrogen concentrations is as follows: Take 2.5g of sodium succinate, 2.5g of sodium citrate dihydrate, 1.0g of K₂HPO₄, 1.0g of KH₂PO₄, and MgSO₄·7H₂O. 0.2g was dissolved in 1000mL of water. Different treatments were prepared by adding 0.471g, 0.942g, 1.884g, 3.297g, 4.710g, and 7.065g of (NH4)2SO4, respectively, which resulted in ammonia nitrogen concentrations of 100mg / L, 200mg / L, 400mg / L, 700mg / L, 1000mg / L, and 1500mg / L. The pH was adjusted to 7.4, and the mixture was sterilized at 121℃ under high temperature and high pressure for 20min. After cooling, 1% (v / v) of sterile filtered composite carbon source mixture, 0.2% (v / v) of composite vitamin mixture, and 0.2% (v / v) of composite trace element mixture were added and mixed well.

[0036] (2) Assessment of tolerance to ammonia nitrogen

[0037] Heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 were inoculated into simulated wastewater with different ammonia nitrogen concentrations at an inoculation rate of 1% v / v. The cultures were then incubated at 28°C and 180 rpm for 48 h. Samples were then taken to measure the OD of the culture medium. 600 The ammonia nitrogen concentration was measured (using Sodium's reagent spectrophotometry) and converted into ammonia nitrogen removal rate. The results are shown in [Figure number missing]. Figure 5 .Depend on Figure 5 It can be seen that the heterotrophic nitrifying bacteria ZFS1-13 has a strong tolerance to simulated wastewater with high ammonia nitrogen. After 48 hours of treatment, the OD in simulated wastewater with an ammonia nitrogen concentration of 1500 mg / L was significantly reduced. 600 The value reached 0.85, and the ammonia nitrogen removal rate was 12.8%.

[0038] Example 4: Application of heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 in a culture medium with ammonia nitrogen as the sole nitrogen source.

[0039] (1) Solution preparation

[0040] Preparation of culture medium with ammonium ions as the sole nitrogen source: Referring to the preparation method of simulated wastewater in Example 3 above, the amount of (NH4)2SO4 added in the simulated wastewater preparation process of Example 3 was adjusted to 0.707 g / L, that is, the ammonia nitrogen concentration was 150 mg / L, and the other components and their concentrations were the same.

[0041] (2) Heterotrophic nitrifying bacteria (Sphingobacterium sp.) ZFS1-13 were inoculated into a culture medium with ammonium ions as the sole nitrogen source at an inoculation rate of 1% v / v. The culture was incubated at 28℃ and 180 rpm. Samples were taken at 0h, 6h, 12h, 24h, 36h, 48h, and 72h to determine the OD of the culture medium. 600 The values ​​were measured, and the concentrations of ammonia nitrogen (Nathaniel's reagent spectrophotometry), nitrite nitrogen (4-aminobenzenesulfonamide spectrophotometry), and nitrate nitrogen (UV spectrophotometry) were determined. The results are shown in [Figure number missing]. Figure 6 .Depend on Figure 6 It can be seen that the heterotrophic nitrifying bacteria ZFS1-13 has a good removal effect on ammonia nitrogen in water. When the bacteria grow for 48 hours, it can remove more than 60% of the ammonia nitrogen in the water with an initial concentration of 150 mg / L.

[0042] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Guangdong Bowo Biotechnology Co., Ltd. <120> A high ammonia nitrogen-tolerant heterotrophic nitrifying bacterium and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1400 <212> DNA <213> Heterotrophic nitrifying bacteria ZFS1-13 (Sphingobacterium sp. ZFS1-13) <400> 1 tgcagtcgac gggatccgcc ggcgagcttg ctcgaaggcg gtgagagtgg cgcacgggtg 60 cgtaacgcgt gagcaacctg cccatatcag gggatagcc cggcgaaagt cggattaaca 120 ccgcatgaca ataacttacc gcatggtaat ttatttaat atttagga tatggatggg 180 ctcgcgtgac attagctagt tggtcggggt aacggcccac caggctacg atgtcttaggg 240 gctctgagag gagaatcccc cacactggta ctgagacacg gaccagactc ctacgggagg 300 cagcagtaag gatatttggt caatgggcgg tagcctgac cagccatgcc gcgtgcagga 360 tgactgccct atgggttgta aactgctttt gtacgggaac aaccgyctgc tcgtgagcag 420 gcctgagtgt accgtaagaa taaggatcgg ctaactccgt gccagcagcc gcggtaatac 480 ggaggatccg agcgttatcc ggatttattg gtttaaagg gtgcgtaggc ggccgcttaa 540 gtcaggagtg aaatacggca gctcactgt cgcagtgctt ttgatactga gtggcttgaa 600 tccatttgaa gtgggcggaa caacaagt agcggtgaa tgcatagata tgtcttagaa 660 ccccgattgc gaaggcagct cactaagctg gattgacgc tgatgcacga aagcgtgggg 720 atcgaacagg attagatacc ctggtagtcc acgccctaaa cgatgatgac tcgatgttag 780 cgatataccg ttagcgtcca agcgaaagcg ttaagtcatc cacctgggga gtacgcccgc 840 aagggtgaaa ctcaaaggaa ttgacgggg cccgcacaag cggaggagca tgtggtttaa 900 ttcgatgata cgcgaggaac cttacccggg cttgaaagtt actgcattat ccagagatgg 960 ataagtcctt cgggacagga aactaggtgc tgcatggctg tcgtcagctc gtgccgtgag 1020 gtgttgggtt aagtcccgca acgagcgcaa cccctatgtt tagttgccag catgtaaagg 1080 tggggactct aaacagactg cctgcgcaag cagtgaggaa ggcggggacg acgtcaagtc 1140 atcatggccc ttacgtccgg ggctacacac gtgctacaat ggacggtaca gcgggcagct 1200 acacagcaat gtggtgcgaa tcccgaaaag ccgttcacag ttcggatcgg ggtctgcaac 1260 tcgaccccgt gaagttggat tcgctagtaa tcgcgtatca gcaatgacgc ggtgaatacg 1320 ttcccgggcc ttgtacacac cgcccgtcaa gccatgaaag ctgggggtgc ctaaagcatg 1380 gccgcaagag cggccagcat 1400

Claims

1. Heterotrophic nitrifying bacteria ( Sphingobacterium sp.) ZFS1-13, its accession number is: GDMCC No: 60528.

2. A heterotrophic nitrifying bacteria microbial inoculant, characterized in that, The microbial agent contains the heterotrophic nitrifying bacteria as described in claim 1. Sphingobacterium sp.) ZFS1-13.

3. The heterotrophic nitrifying bacteria as described in claim 1 ( Sphingobacterium sp.) Z The application of heterotrophic nitrifying bacteria microbial agents as described in FS1-13 or claim 2 in the removal of ammonia nitrogen.

4. The application according to claim 3, characterized in that, It is used in the removal of ammonia nitrogen from water bodies.

5. The application according to claim 3, characterized in that, This is an application for removing ammonia nitrogen from wastewater that can tolerate high concentrations of ammonia nitrogen; the concentration of ammonia nitrogen in the high-concentration ammonia nitrogen wastewater is 1500 mg / L.

6. The application according to claim 3, characterized in that, Heterotrophic nitrifying bacteria ( Sphingobacterium sp.) Z FS1-13 Application of ammonia nitrogen as the sole nitrogen source in ammonia nitrogen removal.

Images