Novel high-efficiency flocculating denitrifying bacteria and application thereof

Abstract

The application discloses a novel high-efficiency flocculation denitrifying bacteria and application thereof. The bacteria is Thauera sp. JM12B12, which is separated and screened from water in a South American white prawn breeding pond, and is preserved in the Guangdong Microbial Digital Culture Collection Center (GDMCC) on June 27, 2023, and the address is No. 59 Building, Yard, 100, Middle Martyrs Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation number is GDMCC No. 63587. The Thauera sp. JM12B12 of the application is separated from water in a South American white prawn breeding pond, that is, the removal rates of nitrate nitrogen and nitrite nitrogen of the strain are both 100% when the carbon-nitrogen ratio is 5-20. In addition, the strain also has a high-efficiency flocculation function, that is, the flocculation efficiencies of the bacterial cells and supernatant are 90.6% and 90.1% respectively when nitrate nitrogen is removed. The novel high-efficiency flocculation denitrifying bacteria JM12B12 disclosed by the application has important application value in removal of denitrification and solid suspended matter of wastewater with excessive nitrogen content, and provides a novel microbial material for water environment protection.

Description

Technical Field

[0001] This invention belongs to the field of microbial and environmental engineering technology, specifically relating to a novel, highly efficient flocculating denitrifying bacteria and its applications. Technical Background

[0002] With the rapid development of China's aquaculture industry, high-density intensive farming models are becoming increasingly popular. As aquaculture shifts from traditional extensive to high-density intensive farming, the pollution of aquaculture water bodies is becoming increasingly prominent. Excessive uneaten feed and the accumulation of large amounts of animal feces and metabolites lead to excessive inorganic nitrogen concentrations and large amounts of suspended solids in the aquaculture water, severely deteriorating its physicochemical properties and ecological environment. This not only harms the healthy growth of farmed animals, affects aquaculture yields, and increases farming costs, but also causes serious pollution to the surrounding ecological environment due to the indiscriminate discharge of untreated aquaculture wastewater. Therefore, exploring efficient and safe measures to restore aquaculture water environments and constructing healthy ecological aquaculture technologies are crucial for the sustainable development of my country's aquaculture industry.

[0003] Wastewater denitrification mainly employs three methods: physical, chemical, and biological. Among these, microbial denitrification technology overcomes some of the shortcomings of physical and chemical denitrification processes, offering advantages such as simple operation, wide applicability, good treatment effect, and virtually no secondary pollution. Therefore, microbial denitrification technology has become the most widely used denitrification technology both domestically and internationally. Currently, environmental engineering primarily utilizes denitrification processes such as anoxic-aerobic, anaerobic-anoxic-aerobic, intermittent, and aerated biological filters. These processes generally separate nitrification and denitrification processes into stages or implement them in different reactors.

[0004] In recent years, researchers have discovered that certain bacterial strains can perform denitrification under aerobic conditions; these bacteria are called aerobic denitrifying bacteria. Most aerobic denitrifying bacteria also possess heterotrophic nitrification capabilities. Therefore, the discovery of aerobic denitrifying bacteria has greatly promoted the development and application of simultaneous nitrification and denitrification processes. Compared with traditional biological nitrogen removal processes, the simultaneous nitrification and denitrification process developed based on aerobic denitrifying bacteria not only achieves simultaneous nitrification and denitrification within an ecosystem or reactor, but also features a fast system reaction rate, short hydraulic retention time, and low energy consumption during nitrogen removal. Aerobic denitrifying bacteria show promising application prospects in nitrogen removal from aquaculture wastewater.

[0005] Microbial flocculants are active substances with certain flocculation properties obtained from microbial cells or metabolites. They have good properties and advantages such as wide applicability, good purification effect, non-toxicity, harmlessness, and biodegradability. They are widely used in the treatment of ecological and environmental pollution such as aquaculture wastewater, livestock and poultry manure, industrial wastewater, and domestic sewage.

[0006] Aerobic denitrifying bacteria and flocculants exhibit physiological, biochemical, and phylogenetic diversity. The aerobic denitrifying bacteria reported in the literature mainly include genera such as *Pseudomonas*, *Paracoccus*, *Alcaligenes*, and *Bacillus*; while the flocculants mainly include *Bacillus*, *Klebsiella*, and *Sphingomonas*. Currently, there are numerous literature reports and national invention patents related to aerobic denitrifying bacteria; however, research on aerobic denitrifying bacteria that exhibit both high denitrification efficiency and high flocculation efficiency is scarce. Summary of the Invention

[0007] The first objective of this invention is to provide a novel and highly efficient flocculating denitrifying bacterium—Thauera sp. JM12B12, which was isolated and screened from the water of Litopenaeus vannamei ponds and deposited on June 27, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No. 63587.

[0008] The Thaurea sp. JM12B12 has the following characteristics:

[0009] 1. Morphological characteristics: Thaurea sp. JM12B12 is a Gram-negative bacterium. When cultured on LB medium at 30°C for 72 hours, the colonies are off-white with regular, round edges, smooth, and opaque surfaces, and the colony diameter is 1-2 mm. The cells are rod-shaped, measuring (0.9 × 2.7) μm, and have a single extreme flagellum.

[0010] 2.16S rRNA characteristics: The 16S rRNA sequence of Thaurea sp. JM12B12 is shown in SEQ ID NO.1. BLAST alignment analysis showed that the 16S rRNA gene sequence of this bacterium had the highest similarity (99.18%) with T. chlorobenzoica 3CB-1, followed by T. selenatis ATCC 55363, T. aminoaromatica S2, T. phenylacetica B4P, and T. mechernichensis TL1, with similarities of 99.17%, 99.11%, 99.04%, and 98.90%, respectively. The similarity with other strains was less than 98.6%.

[0011] 3. Genomic Characteristics: The genome of Thaurea sp. JM12B12 contains 58 contigs, with a size of 4,147,635 bp and a G+C content of 68.0%. The average nucleotide similarity (ANI) and DNA-DNA hybridization value (dDDH) of this bacterium compared to other Thaurea species are both below the threshold for species classification, indicating that strain JM12B12 is a novel species within the genus Thaurea. Furthermore, genomic functional analysis revealed that strain JM12B12 possesses all the key genes required for the conversion of nitrate and / or nitrite nitrogen to nitrogen gas, including the key genes napA (periplasmic nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase), and nosZ (nitrous oxide reductase).

[0012] The second objective of this invention is to provide an application of Thaurea sp. JM12B12 for the removal of nitrate and / or nitrite nitrogen from water. The strain of this invention can efficiently and rapidly remove nitrate and nitrite nitrogen from water; that is, after 18 hours of cultivation, the strain achieves a 100% removal rate for both nitrate and nitrite nitrogen, with no generation or accumulation of nitrite nitrogen during the nitrate removal process, resulting in no secondary pollution. Furthermore, Thaurea sp. JM12B12 achieves a 100% removal rate for either nitrate or nitrite nitrogen at a carbon-to-nitrogen ratio of 5-20. This demonstrates that strain JM12B12 can be applied to denitrification treatment of water bodies under different carbon-to-nitrogen ratio conditions.

[0013] A third objective of this invention is to provide the application of Thaurea sp. JM12B12 in the preparation of flocculants. When removing nitrate nitrogen, the strain of this invention exhibits flocculation efficiencies of 90.1% for its cells and 90.6% for its supernatant in the removal of kaolin suspension. This demonstrates that strain JM12B12 can be applied to the removal of suspended solids in water.

[0014] The fourth objective of this invention is to provide a live bacteria preparation with highly efficient denitrification and flocculation functions, which contains Thaurea sp. JM12B12 as an active ingredient.

[0015] The Thaurasp. JM12B12 strain of this invention was isolated from water from Litopenaeus vannamei ponds. This strain achieved a 100% removal rate of both nitrate and nitrite nitrogen at a carbon-to-nitrogen ratio of 5-20. Furthermore, the strain also exhibits highly efficient flocculation capabilities; specifically, during nitrate nitrogen removal, the flocculation efficiency of its bacterial cells and supernatant were 90.6% and 90.1%, respectively. This novel, highly efficient flocculating denitrifying bacterium, JM12B12, has significant application value in denitrification and solid suspension removal from wastewater with excessive nitrogen content, providing a novel microbial material for water environmental protection.

[0016] Thaurea sp. JM12B12 was isolated and screened from the water of Litopenaeus vannamei ponds and deposited on June 27, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No. 63587. Attached Figure Description

[0017] Figure 1 Colony morphology of Thaurae sp. JM12B12

[0018] Figure 2 Cell morphology of Thaurasa sp. JM12B12 under transmission electron microscopy

[0019] Figure 3 The growth and denitrification characteristics of Thaurea sp. JM12B12 under conditions where nitrate nitrogen is the sole nitrogen source.

[0020] Figure 4 The growth and denitrification characteristics of Thaurea sp. JM12B12 under conditions where nitrite nitrogen is the sole nitrogen source.

[0021] Figure 5 The flocculation effect of Thaurea sp. JM12B12 on bacterial cells and supernatant during nitrate removal.

[0022] Figure 6 The denitrification effect of Thaurera sp. JM12B12 under different carbon-nitrogen ratios. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1: Isolation, purification and preservation of Thaura sp. JM12B12

[0025] Samples were collected from the Litopenaeus vannamei (whiteleg shrimp) aquaculture ponds at the Nianfeng Company's Guanghai Town aquaculture base (N21°56′31″; E112°46′16″) in Taishan City, Jiangmen City, Guangdong Province. 1 mL of aquaculture water sample was serially diluted 10⁻⁶ times. -1 10 -2 10 -3 and 10 -4 Then, take 100 μL of 10 -2 10 -3 and 10 -4The diluted solution was spread onto selective medium used for the isolation and screening of denitrifying bacteria and incubated at 30°C. Colony morphology was observed visually, and single colonies showing morphological differences were picked, streaked, and purified. The purified colonies were inoculated into 5 mL of R2A liquid culture medium and incubated at 30°C and 180 rpm for subsequent storage in glycerol tubes. The strain JM12B12 was thus isolated.

[0026] The selective culture medium formula is as follows: sodium succinate 0.25g, sodium citrate dihydrate 0.25g, NaNO₂ 0.069g, KNO₃ 0.101g, (NH₄)₂SO₄ 0.066g, Na₂HPO₄ 1.0g, KH₂PO₄ 1.0g, MgSO₄·7H₂O 0.2g, 15g agar, pH 7.2-7.4, adjusted to 1000mL, autoclaved at 121℃ for 15min. During plate preparation, a mixture of 1% (v / v) composite carbon source and 0.2% (v / v) trace element solution is added. Composite carbon source: 13.8g D-glucose, 13.8g D-fructose, 13.8g D-lactose, 12.8mL 90% lactic acid, 14.0g mannitol, 14.0mL anhydrous ethanol, 12.6mL glycerol, 9.6g sodium benzoate, 9.2g salicylic acid, 19.0g anhydrous sodium acetate, dissolved in 1000mL water, pH 7.2, sterilized by filtration through a 0.22μm filter membrane. Trace element mixture: EDTA-Na 10.0g, ZnSO4·7H2O 0.5g, CaCl2 5.5g, MnCl2·4H2O 0.4g, FeSO4·7H2O 1.1g, NaMoO4·2H2O 0.4g, CuSO4·5H2O 0.2g, CoCl2·6H2O 0.5g, pH adjusted to 6.0, volume brought to 1000mL, filtered through a 0.22μm filter membrane.

[0027] Example 2: Morphological characteristics of Thaura sp. JM12B12

[0028] like Figure 1 and Figure 2 As shown, strain JM12B12 is a Gram-negative bacterium. When cultured on LB medium for 72 hours, its colony morphology is as follows: off-white, with regular edges, round shape, smooth and opaque surface, and a colony diameter of 1-2 mm. Transmission electron microscopy reveals its cell morphology to be (0.9 × 2.7) μm in size, rod-shaped, with a single extreme flagellum.

[0029] Example 3: 16S rRNA sequence analysis of Thaurasa sp. JM12B12

[0030] Genomic DNA was extracted from strain JM12B12 using a bacterial DNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.). PCR products were amplified using the universal primers 27F / 1492R for bacterial 16S rRNA gene amplification and then sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. Homology analysis of the sequencing results with 16S rRNA gene sequences in the EzBioCloud database showed that the 16S rRNA gene sequence of Thauras sp. JM12B12 had the highest similarity (99.18%) to *T. chlorobenzoica* 3CB-1, followed by *T. selenatis* ATCC 55363, *T. aminoaromatica* S2, *T. phenylacetica* B4P, and *T. mechernichensis* TL1, with similarities of 99.17%, 99.11%, 99.04%, and 98.90%, respectively. The similarity to other strains was below 98.6%. Based on these results, the strain of this invention was preliminarily identified as *Thauera*.

[0031] Example 4: Genomic sequence analysis of Thaurasa sp. JM12B12

[0032] Genomic DNA was extracted from strain JM12B12 and sent to Shanghai Meiji Biotechnology Co., Ltd. for genome sequencing. The sequencing results were then used to assemble the genome and assess its quality using SPAdes v3.11.1 and CheckM 1.0.9, respectively. The mean nucleotide similarity (ANI) between JM12B12 and other model strains with a 16S rRNA gene sequence similarity greater than 98.65% was calculated using fastANI. The DNA-DNA hybridization value (dDDH) between JM12B12 and other model strains was calculated using the Genome-to-Genome Distance Calculator on the DSMZ website. The results are shown in Table 1. The ANI values ​​of strain JM12B12 and other model bacteria ranged from 84.2% to 87.7%, and the dDDH values ​​ranged from 26.3% to 31.4%, both values ​​being lower than the bacterial species-level classification thresholds (ANI threshold of 95% and dDDH threshold of 70%). The above results confirm that strain JM12B12 is a new species of the genus Thaurea.

[0033] Table 1. ANI and dDDH values ​​of strain JM12B12 compared with other model strains

[0034]

[0035]

[0036] The strain JM12B12 was named Thaurea sp. JM12B12 and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit date was June 27, 2023, and the accession number was GDMCC 63587.

[0037] Example 5: Denitrification and High-Efficiency Flocculation Characteristics of Thaurasp. JM12B12

[0038] After inoculating strain JM12B12 into LB liquid medium and culturing for 24 h, centrifuging at 6000 rpm for 10 min, discarding the supernatant, washing twice with sterile physiological saline, and resuspending, adjusting the OD of the bacterial suspension. 600 Approximately 1.0. The bacterial suspension was inoculated at a 4% inoculum (v / v) into a solution containing nitrate nitrogen (NO3). - -N) and nitrite nitrogen (NO2) - In a culture medium where NO3- is the sole nitrogen source, - -N and NO2 - The initial concentration of -N was set to 80 mg / L. Samples were taken at 0, 12, 18, 24, and 48 h of culture. A portion of the culture medium was used to detect OD. 600 The other part of the culture medium was centrifuged at 8000 for 5 min, and the supernatant was collected for NO3 determination. - -N and NO2 - -N concentration. NO3 - -N was determined by ultraviolet spectrophotometry. NO2 - -N was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry.

[0039] strain JM12B12 is undergoing NO3-- -After culturing in a medium with -N as the sole nitrogen source for 48 hours, the flocculation efficiency of bacterial cells (resuspended in an equal volume of sterile water after centrifugation) and sterile supernatant was measured. The experimental group's flocculation measurement system (40 mL) consisted of 2 mL of bacterial suspension or 2 mL of supernatant, 2 mL of CaCl2 (1%, w / v), and 36 mL of kaolin (5 g / L), placed in a 100 mL beaker. 1% NaOH or HCl was added to adjust the pH to approximately 7.5. The blank control group's flocculation system (40 mL) used sterile water and blank culture medium instead of bacterial suspension and supernatant, respectively, with the remaining components and amounts identical to the experimental group. The flocculation mixture was stirred in two stages using a magnetic stirrer: rapid stirring at 500 rpm for 1 min, and slow stirring at 100 rpm for 5 min. After stirring, let the mixture stand at room temperature for 5 minutes. Then, use a pipette to aspirate the liquid from 1 cm below the surface and measure its absorbance at 550 nm. The formula for calculating the flocculation efficiency is as follows:

[0040] Flocculation efficiency (%) = (AB) / A × 100

[0041] A and B represent the absorbance at 550 nm for the blank control group and the experimental group, respectively.

[0042] With NO3 - Culture medium with -N as the sole nitrogen source: Sodium lactate 1.3 mL, KNO3 0.57 g, Na2HPO4 1.6 g, KH2PO4 1.0 g, MgCl2·6H2O 0.1 g, Vitamin B12 5 mg, Biotin 10 mg, with 0.2% trace element mixture added (v / v), pH 7.2, adjusted to 1000 mL, autoclaved at 115 °C for 20 min. (NO2 is used as the nitrogen source.) - Medium with -N as the sole nitrogen source: 1.3 mL sodium lactate, 0.39 g NaNO2, 1.6 g Na2HPO4, 1.0 g KH2PO4, 0.1 g MgCl2·6H2O, 5 mg vitamin B12, 10 mg biotin, with 0.2% trace element mixture added, pH 7.2, bring to a final volume of 1000 mL, autoclave at 115 °C for 20 min.

[0043] As attached Figure 3 As shown, strain JM12B12 has an effect on NO3- in water. - -N has a good removal effect, that is, after 18 hours of culture, the OD of the bacterial culture is reduced. 600 It is 0.6 for NO3. - The removal efficiency of -N has reached 100%, and no NO2 is generated during the denitrification process. - -N is generated and accumulated. (See attached image.) Figure 4 As shown, strain JM12B12 has an effect on NO2 in water.- -N also showed good removal efficiency, that is, after 18 hours of cultivation, the OD of the bacterial culture was [value missing]. 600 The value is 0.3 for NO2. - The removal efficiency of -N reached 100%. The above results indicate that Thaura sp. JM12B12 is an environmentally friendly and highly efficient denitrifying strain with great application potential in water denitrification treatment.

[0044] As attached Figure 5 As shown, strain JM12B12 exhibited flocculation efficiencies of 90.1% for cell culture and 90.6% for supernatant during nitrate removal. This indicates that strain JM12B12 cells and their secreted extracellular metabolites possess the potential to act as highly efficient microbial flocculants, and this bacterium has significant application value in the removal of suspended solids from wastewater.

[0045] Example 6: Denitrification effect of Thaurasa sp. JM12B12 under different carbon-nitrogen ratios

[0046] JM12B12 bacterial suspension was prepared according to the method in Example 5. The bacterial suspension was inoculated at a 4% inoculum (v / v) onto a substrate containing NO3. - -N, NO2 - In a denitrified culture medium where nitrogen (N) was the sole nitrogen source and sodium lactate was the sole carbon source, the amount of sodium lactate added was adjusted to achieve carbon-to-nitrogen ratios (molar ratios) of 1, 2, 5, 8, 10, 15, 18, and 20. After inoculation, the culture medium was placed at 30°C and incubated statically for 48 hours. The NO3- content in the supernatant was then measured. - -N and NO2 - -N concentration.

[0047] As attached Figure 6 As shown, strain JM12B12 exhibits resistance to NO3- at a carbon-to-nitrogen ratio of 5-20. - -N and NO2 - The removal rate of -N reached 100%, indicating that strain JM12B12 has a high efficiency of denitrification and nitrogen removal under both low and high carbon-nitrogen ratios. This means that the strain has important application value in the denitrification treatment of wastewater polluted by oligotrophic or eutrophic nitrogen.

[0048] 16S rRNA nucleotide sequence listing (SEQ ID NO.1)

[0049] GAACTTAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAACGGCAGCGGGGGCTTCGGCCTGCCGGCGAGTGGCGAACGGGTGAGTAATGCATCGGAACGTGCCCATGTCGTGGGGGATAACGTAGCGAAAGCTACGCTAATACCGCATACGTCCTGAGGGAGAAAGCGGGGGATCTTCGGACCTCGCGCGATTGGAGCGGCCGATGTCGGATTAGCTAGTAGGTGGGGTAAAGGCCCACCTAGGCGACGATCCGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCGGCCGGGAAGAAATCGCACGCTCTAACATAGCGTGTGGATGACGGTACCGGACTAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGACAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCGTTTGTGACTGCAAGGCTAGAGTACGGCAGAGGGGGGTGGAATTCCTGGTGTAGCAGTGAAATGCGTAGATATCAGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGCCTGTACTGACGCTCATGCAC

[0050] GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTC

[0051] GACTAGTCGTTCGGAGCAGCAATGCACTGAGTGACGCAGCTAACGCGTGAAGTCGACC

[0052] GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGACCCGCACAA

[0053] GCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATG

[0054] TCTGGAACCTTGCTGAGAGGCGAGGGTGCCTTCGGGAGCCAGAACACAGGTGCTGCAT

[0055] GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT

[0056] TGTCACTAGTTGCCATCATTTGGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGG

[0057] AGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCA

[0058] TACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCCAATCCCTTAAAGCC

[0059] GATCGTAGTCCGGATCGTAGTCTGCAACTCGACTACGTGAAGTCGGAATCGCTAGTAATC

[0060] GCAGATCAGCATGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACAC

[0061] CATGGGAGTGGGTTTCACCAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACG

[0062] GTGAGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTG

[0063] GATCACCTCCTTT

Claims

1. Taurella multocida ( Thauera sp.) JM12B12, accession number GDMCC No. 63587.

2. The application of the *Taurizobacterium* JM12B12 as described in claim 1 in the removal of nitrate nitrogen and / or nitrite nitrogen from water.

3. The application according to claim 2, characterized in that, The carbon-to-nitrogen ratio of the water body is 5-20.

4. The application of the *Taurizobacterium* JM12B12 as described in claim 1 in the preparation of flocculants.

5. A live bacteria preparation with denitrification or flocculation functions, characterized in that, It contains the Taureella JM12B12 as described in claim 1.

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