Salt-tolerant denitrifying bacterium and application thereof

Abstract

The invention discloses a salt-tolerant denitrifying bacterium and application of the salt-tolerant denitrifying bacterium, the salt-tolerant denitrifying bacterium is specifically Stutzerimonas degrandans and is named as Stutzerimonas degrandans BP, the preservation number of the salt-tolerant denitrifying bacterium is GDMCC No.64297, and the salt-tolerant denitrifying bacterium is preserved in the Guangdong Microbial Culture Collection Center on January 19, 2024. When the Stutzerimonas degrandans BP provided by the invention is used for biological nitrogen removal of high-salinity wastewater, the Stutzerimonas degrandans BP has relatively strong salt-tolerant denitrification capability, NO3 <->-N in a high-salinity water body can be effectively removed, no by-products such as NO2 <->-N and NH4 < + >-N are accumulated, and secondary pollution cannot be caused. Experimental results show that the Stutzerimonas degrandans BP provided by the invention is a facultative anaerobic bacterium, and is suitable for biological nitrogen removal in an anaerobic or anoxic environment. Under an anaerobic condition, high-efficiency salt-tolerant denitrification can be realized by virtue of relatively small biomass (OD600 = 0.15-0.25), so that the yield of sludge can be reduced while the operation cost of the process is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to a salt-tolerant denitrifying bacterium and application thereof. Background Art

[0003] At present, the industry often uses ion exchange, evaporation crystallization, reverse osmosis and catalytic reduction to treat high-salinity nitrate nitrogen wastewater, but these methods have problems such as pollution transfer and secondary pollution, and cannot achieve the final disposal of pollutants. Compared with the above methods, biological denitrification has the advantages of low operating cost, high efficiency and stability, and no secondary pollution.

[0004] Traditional denitrifying bacteria can convert NO in wastewater into 3 - -N Restore to N x O and N 2 It is removed from wastewater and plays an important role in biological denitrification of wastewater. However, the appearance of high-salt environment will cause a sharp increase in its cell osmotic pressure, affect the normal structure of the cell, and even cause the death of microorganisms, inhibit its metabolic capacity and enzyme activity, and cause a large accumulation of intermediate by-products. The efficiency of biological denitrification is greatly reduced and it is difficult to meet the discharge requirements. Therefore, screening salt-tolerant and efficient denitrifying bacteria is one of the effective ways to solve the denitrification of high-salt wastewater.

[0005] Halophilic bacteria rely on K uptake + exclude + , intracellular accumulation of compatible solutes and other salt-tolerant regulatory mechanisms balance the osmotic pressure inside and outside the cell, maintain the activity of enzymes, and thus complete growth and metabolism in a high-salt environment. Studies have shown that the use of salt-tolerant denitrifying bacteria for biological denitrification can achieve efficient nitrogen removal in a high-salt environment. Therefore, it is urgent to develop a denitrifying bacteria source with excellent salinity tolerance for biological denitrification in a high-salt environment.

[0006] In the past, salt-tolerant denitrifying bacteria usually required a high biomass to achieve good nitrate removal. Studies have shown that for 150 mg / L nitrate, the biomass range is generally between 0.51.5 (OD 600 ), which not only increases the volume and operating costs of the bioreactor, but also leads to an increase in sludge production, increasing the burden of subsequent treatment and disposal. To this end, it is necessary to develop new denitrifying bacteria sources to reduce the biomass demand for denitrification, reduce the volume of the bioreactor, save space and construction costs, reduce the cost of cultivating and maintaining microorganisms, reduce sludge production, and reduce the pressure of subsequent treatment. At the same time, it can increase the denitrification load of microorganisms, which is conducive to further expanding the application scope and applicable scenarios of bacteria. Summary of the invention

[0007] In view of the problems existing in the above-mentioned prior art, the object of the present invention is to provide a salt-tolerant denitrifying bacterium and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a salt-tolerant denitrifying bacterium, which is Stutzerimonas degradans, named Stutzerimonas degradans BP, with a preservation number of GDMCC No: 64297, and was deposited in Guangdong Provincial Microbial Culture Collection Center on January 19, 2024.

[0010] The 16S rDNA gene sequence determined by the Stutzerimonas degradans BP is shown in SEQ ID NO.1.

[0011] The second aspect of the present invention provides a method for culturing the above-mentioned salt-tolerant denitrifying bacteria, comprising the following steps:

[0012] S1. Inoculate a single colony of pure salt-tolerant bacteria into the seed culture medium and culture under aerobic conditions. Control the culture temperature at 30℃~35℃ and shake culture at 130~150rpm for 20~24h to achieve large-scale proliferation. Cultivate to the late logarithmic phase, centrifuge the bacterial solution, resuspend it in PBS, and prepare the OD 600 It is a bacterial suspension of 1.8 to 2.2;

[0013] S2. Use denitrification medium to screen denitrifying bacteria, inoculate 4.5% to 5.5% bacterial suspension, and control the initial OD 600 The concentration of anaerobic culture medium is 0.095-0.105, and anaerobic static culture is carried out at 30-38℃ for 2-4 days.

[0014] Preferably, the seed liquid culture medium composition is: 10 g·L -1 Yeast extract powder, 7.5g·L -1 Peptone, 20 g L - 1 MgSO 4 7H 2 O, 3.42 g·L -1 C 6 H 5 Na 3 O 7 ·2H 2 O, 2g·L -1 KCl, 0.05 g L -1 FeSO 4 7H 2 O, 40g·L -1 NaCl.

[0015] Preferably, the denitrification culture medium composition is: 1.08 g·L -1 KNO 3 , 4.1 g·L -1 CH 3 COONa, 6.26 g·L - 1 K 2 HPO 4 ·3H 2 O, 1.24 g·L -1 KH 2 PO 4 , 0.2g·L -1 MgSO 4 , 40g·L -1 NaCl.

[0016] The third aspect of the present invention provides the use of the above-mentioned salt-tolerant denitrifying bacteria in denitrification under salinity stress.

[0017] Preferably, the salinity ranges from 0 to 6%, and more preferably from 0 to 4%.

[0018] Preferably, the biomass OD of the salt-tolerant denitrifying bacteria under salinity stress is 600 It is 0.15~0.25.

[0019] The Stutzerimonas degradans BP screened by the present invention has the following morphological characteristics: after solid culture for 48 hours, the colony is light brown, round, slightly raised, smooth on the surface, and has complete edges; Gram staining is negative; the bacteria are rod-shaped, with blunt ends, and the size is (3μm-4μm)×(8μm-14μm). Its physiological and biochemical characteristics are: facultative anaerobic, and the suitable salinity is 0-6%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The Stutzerimonas degradans BP provided by the present invention has a strong salt-tolerant denitrification ability and can efficiently remove NO in high-salinity water bodies. 3 - -N, and no byproduct NO 2 - -N and NH 4 + -N accumulation, will not cause secondary pollution; small biomass (OD 600=0.15~0.25) can realize efficient salt-tolerant denitrification, thereby reducing the process operation cost and improving the biological utilization rate. Therefore, it has broad application prospects in the biological denitrification of high-salt wastewater.

[0022] (2) The Stutzerimonas degradans BP provided by the present invention is a facultative anaerobic bacterium that can reproduce in large quantities under aerobic conditions, providing a large amount of biological reserves for subsequent denitrification, has strong environmental adaptability, and is suitable for biological denitrification in anaerobic or anoxic environments.

[0023] (3) Compared with the traditional activated sludge method, the biological denitrification treatment using the Stutzerimonas degradans BP provided by the present invention can also reduce the generation of sludge, which helps to reduce the cost of subsequent sludge treatment and disposal.

[0024] Collection Information

[0025] Preservation time: January 19, 2024;

[0026] Depository: Guangdong Microbiological Culture Collection Center;

[0027] Deposit number: GDMCC No:64297;

[0028] Address of the depository: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou;

[0029] Postal code: 510070;

[0030] Taxonomic nomenclature: Pseudomonas phenolilytica. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is the colony morphology of Stutzerimonas degradans BP;

[0033] Figure 2 This is the Gram staining image of Stutzerimonas degradans BP;

[0034] Figure 3This is a scanning electron micrograph of Stutzerimonas degradans BP;

[0035] Figure 4 This is the phylogenetic tree diagram of Stutzerimonas degradans BP;

[0036] Figure 5 is the OD of Stutzerimonas degradans during BP under different salinity stress in Experiment 1 600 Change graph;

[0037] Figure 6 is the NO in the BP process of Stutzerimonas degradans under different salinity stresses in Experiment 1 3 - -N concentration variation diagram;

[0038] Figure 7 is the NO in the BP process of Stutzerimonas degradans under different salinity stresses in Experiment 1 2 - -N concentration variation diagram;

[0039] Figure 8 This is a graph showing the change in TN concentration during the BP process of Stutzerimonas degradans under different salinity stresses in Experimental Example 1. DETAILED DESCRIPTION

[0040] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0041] Example 1

[0042] 1. Prepare experimental materials

[0043] Enrichment medium: 10 g L -1 Yeast extract powder, 7.5g·L -1 Peptone, 20 g L -1 MgSO 4 7H 2 O, 3.42 g·L -1 C 6 H 5 Na 3 O 7 ·2H 2 O, 2g·L -1 KCl, 0.05 g L -1FeSO 4 7H 2 O, 40g·L -1 NaCl.

[0044] Isolation medium: 10 g L -1 Yeast extract powder, 7.5g·L -1 Peptone, 20 g L -1 MgSO 4 7H 2 O, 3.42 g·L -1 C 6 H 5 Na 3 O 7 ·2H 2 O, 2g·L -1 KCl, 0.05 g L -1 FeSO 4 7H 2 O, 40g·L -1 NaCl, 20 g L -1 Agar.

[0045] Screening medium: 1.08 g·L -1 KNO 3 , 4.1 g·L -1 CH 3 COONa, 6.26 g·L -1 K 2 HPO 4 ·3H 2 O, 1.24 g·L -1 KH 2 PO 4 , 0.2g·L -1 MgSO 4 , 40g·L -1 NaCl.

[0046] Trace elements: 0.1 g·L -1 FeSO 4 7H 2 O, 0.02g·L -1 ZnSO 4 7H 2 O, 0.04g·L -1 CuSO 4 ·5H 2 O, 0.04g·L -1 MnSO 4 ·H 2 O, 0.03g·L -1 H 3 BO 3 , 0.04g·L-1 Na 2 MoO 4 ·2H 2 O, 0.05g·L -1 CoCl 2 6H 2 O.

[0047] Seed culture medium: The composition is the same as the above-mentioned enrichment culture medium.

[0048] Denitrification medium: The composition is the same as the above screening medium.

[0049] 2. Isolation and screening of salt-tolerant denitrifying bacteria

[0050] (1) Preparation of anaerobic vials:

[0051] 30 mL of denitrification culture medium was placed in a 50 mL medical glass infusion bottle, deoxygenated by nitrogen aeration for 15 min, covered with a rubber stopper and sealed with an aluminum cap, and then sterilized in a high-pressure steam autoclave at 121°C for 20 min.

[0052] (2) Isolation and purification of salt-tolerant denitrifying bacteria:

[0053] The bacterial source was the activated sludge from the biochemical treatment unit of an epoxy resin wastewater plant. -1 The activated sludge was diluted with PBS buffer in a gradient manner. 4 , 10 5 , 10 6 100 μL of the bacterial solution was evenly spread on the separation plate and inverted for 48 h at 30°C under aerobic conditions. Colonies with different morphologies were picked and separated and purified by four-zone lines until the colonies and the microscopic bacterial bodies were consistent, and pure salt-tolerant bacteria were obtained.

[0054] (3) Screening of salt-tolerant denitrifying bacteria

[0055] Denitrifying bacteria were screened from the pure salt-tolerant bacteria isolated and purified.

[0056] A single colony of pure salt-tolerant bacteria was inoculated into 50 mL of seed culture medium and cultured under aerobic conditions. The culture temperature was controlled at 30 °C and the shaking culture was carried out at 150 rpm for 24 h to achieve large-scale proliferation. The culture was cultured to the late logarithmic phase (OD 600 =1), centrifuge the bacterial solution and resuspend it in PBS, repeat this process twice to prepare OD 600 2. Denitrifying bacteria were screened using denitrification medium, and 2 mL·L -1 The trace element solution was set to 4% salinity, pH was adjusted to 7, and 5% bacterial suspension was inoculated to control the initial OD 600The concentration of NO was 0.1, and aerobic and anaerobic cultures were performed respectively: (a) aerobic culture was performed by adding 100 mL of the system into a 150 mL conical flask and culturing at 30°C and 150 rpm for 3 days; (b) anaerobic culture was performed by adding 30 mL of the system into a 50 mL medical glass infusion bottle, deoxygenating it with nitrogen, and then culturing it at 30°C for 3 days; after 3 days, NO was measured respectively. 3 - -N, NO 2 - -N, NH 4 + -N and TN content. The strain with the strongest salt tolerance and denitrification ability was selected as the target strain for subsequent experiments.

[0057] After screening, two salt-tolerant bacteria with denitrification ability were found. 3 - -N concentration is 150.58 mg·L -1 Under the conditions of aerobic conditions, strain A had a stronger ability to reduce nitrate than that under anaerobic conditions. After 3 days of cultivation, NO 3 - -N concentration dropped to 5.39 mg·L -1 , but 86.52 mg·L -1 NO 2 - -N and 30.98mg·L -1 Compared with aerobic conditions, strain B had stronger nitrate reduction ability under anaerobic conditions. After 3 days of cultivation, NO 3 - -N concentration dropped to 4.08 mg·L -1 , and there is no NO 2 - -N and NH 4 + -N accumulation, the total nitrogen removal rate reached 96.43%, and the salt-tolerant denitrification was thorough, so strain B was selected as the target strain and subsequent experiments were carried out.

[0058] 2. Identification of salt-tolerant denitrifying bacteria

[0059] (1) Morphological identification

[0060] like Figure 1 As shown in the figure, after strain B was streaked and cultured on a solid separation plate for 48 hours, the colonies were light brown, round, slightly raised, smooth on the surface, and with complete edges. Figure 2 As shown by Figure 2 The results showed that the bacteria were red and identified as Gram-negative bacteria. Figure 3 As shown by Figure 3The results showed that the bacteria were rod-shaped with blunt ends and a size of (3μm~4μm)×(8μm~14μm).

[0061] (2) Molecular Biological Identification

[0062] The genome extraction kit ( The genome of strain B was extracted by using the GenBank Spin Kit for Soil, and the A260 / A280 ratio was 1.8 as determined by a micro-spectrophotometer. The genome was qualified by the quality inspection and used as the DNA template for PCR reaction.

[0063] The universal primers for PCR were: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'); the reaction system was 25 μL, Premix version 2.0 12.5 μL, 27F (10 nM) 1 μL, 1492R (10 nM) 1 μL, DNA template 1 μL, and sterile water was added to 25 μL; the reaction conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 90 s, final extension at 72 °C for 7 min, and 35 cycles.

[0064] After PCR amplification, the PCR amplification product was detected by 1% agarose gel electrophoresis to confirm the PCR amplification fragment, the PCR amplification product was recovered, and the PCR amplification product was subjected to DNA sequencing.

[0065] The obtained DNA sequence is shown in SEQ ID NO: 1. After sequencing, the 16S rDNA sequence of the strain was 1439 bp in length. The spliced ​​sequence was compared with the data in the NCBI database by homology using the NCBI Blast program, and the phylogenetic tree was constructed using the Neighbour Joining method using the Systematic Analysis (MEGA11) software (see Figure 4 ), it was found that the target strain had the closest evolutionary distance to Stutzerimonas degradans, and was determined to belong to Stutzerimonas degradans, and was finally named Stutzerimonas degradans BP.

[0066] Test Example 1

[0067] The obtained Stutzerimonas degradans BP was applied to denitrification under different salinity stresses.

[0068] The specific steps are:

[0069] (1) Inoculate a single colony from the solid plate into a 150 mL conical flask containing 50 mL of seed culture medium and culture at 30 °C with shaking at 150 rpm for 24 h until the late logarithmic phase (OD 600 =1). Use 10mmol·L -1 The seed solution was prepared with phosphate buffer. The bacterial solution was centrifuged at 3500 rpm for 10 min and then resuspended in PBS. This was repeated twice to prepare the OD 600 =2 bacterial suspension.

[0070] (2) The salinity gradient for denitrification was set to 0%, 2%, 4%, 6%, and 8%, and 2 mL·L of trace elements were added. -1 , adjust the pH to 7, dispense 30 mL into a 50 mL anaerobic vial, aerate with nitrogen for 15 min to deoxygenate, inoculate at 5% inoculum, and set the initial biomass OD 600 The concentration of NO was controlled at 0.1, and anaerobic denitrification was carried out under different salinity conditions, and the culture was kept at 30℃ for 3 days. During this period, samples were taken every 12 hours to measure NO 3 - -N,NO 2 - -N, NH 4 + -N, TN, OD 600 . Among them, NO 3 - -N test using UV spectrophotometry, NO 2 - -N was tested by α-naphthylamine hydrochloride spectrophotometry, NH 4 + -N test uses Nessler reagent spectrophotometry, and TN test uses alkaline potassium persulfate digestion UV spectrophotometry. OD within 72h under different salinity environments 600 、NO 3 - -N, NO 2 - -N and TN concentration changes are shown in Figures 5 to 8 shown.

[0071] Depend on Figures 5 to 8 The results show that the denitrification time varies under different salinity conditions. 3 - -N reduction showed a significant positive correlation with the growth of Stutzerimonas degradansBP. Under 0-4% salinity, Stutzerimonas degradansBP could grow normally and remove more than 97% of NO within 72 hours. 3 - -N.

[0072] When the salinity of the culture medium is 0, the low external osmotic pressure causes water to enter the cells, the cell volume expands, and the cell membrane is damaged by excessive pressure, affecting the normal growth and reproduction of bacteria. The bacterial growth is low, but 93.73% of TN can be removed after 24 hours of culture.

[0073] When the salinity in the culture medium increased from 0 to 2%, the bacteria were able to grow normally, overcoming the effect of low osmotic pressure at 0 salinity. The growth of Stutzerimonas degradans BP and NO 3 - -N removal capacity was enhanced, and the bacterial growth in the stable period (OD 600 ) increased from 0.149 to 0.207, 12h NO 3 - The -N reduction rate increased from 52.51% to 92.73%, and the TN removal rate could reach 95.99% within 24 hours.

[0074] When the salinity in the culture medium increased to 4%, the bacteria were inhibited by salinity, the adaptation period of growth increased to 24 hours, the denitrification efficiency decreased, and it took 48 hours of culture to reach a nitrate reduction rate of more than 90%. However, after 72 hours of culture, the TN removal rate could still reach 96.43%.

[0075] In a 0-4% salinity environment, no intermediate NO is present after the reaction is completed. 2 - -N accumulation, but in the reaction process, due to the salinity of NO 2 - -N has a greater inhibitory effect on reduction than NO 3 - -N restore, NO appears 2 - -N accumulation, and the difference between the two increased significantly with the increase of salinity.

[0076] When the salinity increased to 6%, the growth of Stutzerimonas degradans BP was inhibited for up to 60 h, and only 31.9% of NO was reduced in 72 h. 3 - -N.

[0077] When the salinity was further increased to 8%, the strain Stutzerimonas degradans BP still could not achieve growth and NO after 72 h of cultivation. 3 --N removal. This is because the salt concentration at this time has exceeded the threshold that Stutzerimonasdegradans BP can tolerate. The strain cannot resist the external osmotic pressure through self-regulation, which seriously inhibits the growth and metabolism of bacteria.

[0078] In summary, the strain Stutzerimonas degradans BP can grow and perform denitrification in an environment with a salt concentration of 0-6%, especially when the salinity is 0-4%, it can remove more than 97% of NO within 72 hours. 3 - -N, and no NO 2 - -N and NH 4 + -N accumulation. Therefore, Stutzerimonas degradans BP shows a very broad application prospect in the denitrification and denitrification of high-salinity wastewater.

[0079] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention.

Claims

1. A salt-tolerant denitrifying bacterium, characterized in that: The salt-tolerant denitrifying bacteria is Stutzerimonas degradans, named Stutzerimonas degradans BP, with a preservation number of GDMCC No.64297, and was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 19, 2024.

2. A method for culturing salt-tolerant denitrifying bacteria as claimed in claim 1, characterized in that: The steps include: S1. Inoculate a single colony of pure salt-tolerant bacteria into the seed culture medium and culture it under aerobic conditions. Control the culture temperature at 30-35°C and shake culture at 130-150 rpm for 20-24 hours to achieve large-scale proliferation. Cultivate the culture solution to the late logarithmic phase, centrifuge it, resuspend it in PBS, and prepare the OD 600 It is a bacterial suspension of 1.8 to 2.2; S2. Use denitrification medium to screen denitrifying bacteria, inoculate 4.5% to 5.5% bacterial suspension, and control the initial OD 600 The concentration of anaerobic culture medium is 0.095-0.105, and anaerobic static culture is carried out at 30-38℃ for 2-4 days.

3. The method for culturing salt-tolerant denitrifying bacteria according to claim 2, characterized in that: The seed liquid culture medium composition: 10g·L -1 Yeast extract powder, 7.5g·L -1 Peptone, 20 g L -1 MgSO4·7H2O, 3.42 g·L -1 C6H5Na3O7·2H2O、2g·L -1 KCl, 0.05 g L -1 FeSO4·7H2O, 40g·L -1 NaCl.

4. The method for culturing salt-tolerant denitrifying bacteria according to claim 2, characterized in that: The denitrification culture medium composition: 1.08 g·L -1 KNO3, 4.1 g·L -1 CH3COONa, 6.26 g·L -1 K2HPO4·3H2O, 1.24 g·L -1 KH2PO4, 0.2 g·L -1 MgSO4, 40g·L -1 NaCl.

5. Use of the salt-tolerant denitrifying bacteria as claimed in claim 1 for denitrification and denitrification under salinity stress.

6. The use according to claim 5, characterized in that: The salinity ranges from 0 to 6%.

7. The use according to claim 5, characterized in that: The biomass OD of the salt-tolerant denitrifying bacteria under salinity stress 600 It is 0.15~0.25.

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