A Hunan Pseudomonas strain PAD-1, wastewater treatment agent and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a Hunan Pseudomonas strain PAD-1, a wastewater treatment agent and an application thereof. The present invention provides a Hunan Pseudomonas strain PAD-1 (Pseudomonashunanensis strain PAD-1), and the Hunan Pseudomonas strain PAD-1 has a deposit number of CCTCCNO: M20221381. The Hunan Pseudomonas strain PAD-1 of the present invention has aerobic denitrification ability, and also has the advantages of low cost and green environmental protection.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Hunan Pseudomonas strain PAD-1, a wastewater treatment agent and applications thereof. Background Art

[0002] At present, due to human production activities, a large amount of nitrate has entered the ecosystem, leading to eutrophication of lakes and rivers. In order to solve this ecological problem, local governments have built a large number of sewage treatment plants. In these sewage treatment plants, biological denitrification is the most effective measure to remove nitrate. Usually, in order to ensure the high efficiency of biological denitrification, relevant technicians will add additional organic carbon sources to maintain the denitrification activity of the sewage treatment system. However, the addition of organic carbon sources not only increases the operating costs of sewage treatment plants but also causes serious secondary pollution.

[0003] Many studies have reported that Fe(II) can promote the growth of denitrifying bacteria and increase the denitrification rate. Researchers have also observed that the addition of Fe(II) is beneficial to the synthesis of heme and increases the relative abundance of denitrification functional genes, thereby promoting the denitrification rate. However, most of these studies focus on ferrous salts and iron filings, which leads to higher operating costs.

[0004] With the sharp rise in commodity prices today, a low-cost, green and environmentally friendly efficient nitrogen removal technology is needed. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a Hunan Pseudomonas strain PAD-1, a wastewater treatment agent and its application. The Hunan Pseudomonas strain PAD-1 of the present invention has aerobic denitrification ability, and also has the advantages of low cost and green environmental protection.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a Pseudomonas hunanensis strain PAD-1 (Pseudomonas hunanensis strain PAD-1), and the preservation number of the Pseudomonas hunanensis strain PAD-1 is CCTCC NO:M 20221381.

[0008] The present invention provides a wastewater treatment agent, wherein the effective ingredients of the wastewater treatment agent include the Hunan Pseudomonas strain PAD-1 and / or the Hunan Pseudomonas strain PAD-1 preparation described in the above technical solution.

[0009] Preferably, the Hunan Pseudomonas strain PAD-1 preparation includes one or more of Hunan Pseudomonas strain PAD-1 culture, culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant and fermentation broth extract.

[0010] Preferably, the OD of Hunan Pseudomonas strain PAD-1 in the wastewater treatment agent is 600 =0.91~1.63.

[0011] Preferably, the effective ingredient of the wastewater treatment agent also includes pyrite.

[0012] Preferably, the concentration of pyrite in the wastewater treatment agent is 0.8 to 3.2 g / L.

[0013] The present invention provides the use of the Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution in wastewater treatment.

[0014] The present invention provides the use of the Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution in the treatment of nitrogen-rich wastewater.

[0015] The present invention provides a method for treating wastewater, the method comprising the following steps:

[0016] The Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution is mixed with wastewater to treat the wastewater.

[0017] Preferably, the inoculation amount of the Hunan Pseudomonas strain PAD-1 is 0.1% to 1% of the wastewater volume.

[0018] Beneficial effects: The present invention provides a Hunan Pseudomonas strain (Pseudomonas hunanensis strain) PAD-1, and the Hunan Pseudomonas strain PAD-1 has a deposit number of CCTCC NO: M20221381. The Hunan Pseudomonas strain PAD-1 described in the present invention has aerobic denitrification ability, can directly convert nitrate nitrogen into gas products, and has less accumulation of nitrite nitrogen and ammonia nitrogen, further achieving the effect of effectively treating low C / N wastewater, while also having the advantages of low cost and green environmental protection. The strain shows excellent performance in treating low-carbon wastewater, and its cultivation method is simple to operate, with low energy consumption and cost. The Hunan Pseudomonas strain PAD-1 described in the present invention can be made into a new microecological bacterial agent, which has good application prospects in composite wastewater treatment.

[0019] Moreover, the present invention also provides a wastewater treatment agent. The strain described in the present invention can be coupled with pyrite to treat low C / N wastewater. The strain described in the present invention has a stronger denitrification efficiency under the synergistic effect of pyrite, and also has a better carbon source utilization rate. The wastewater treatment agent described in the present invention also has the advantages of being easy to use and simple to operate, thereby reducing the cost of sewage treatment. Therefore, the strain described in the present invention can achieve the effect of effectively treating wastewater, and the strain described in the present invention can be coupled with pyrite to treat low C / N wastewater with a more significant treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 is the colony morphology of strain PAD-1;

[0022] Figure 2 is the phylogenetic tree of strain PAD-1;

[0023] Figure 3 This is the amplification result of nitrogen pathway related genes of Hunan Pseudomonas PAD-1;

[0024] Figure 4 The growth of the strain Hunan Pseudomonas PAD-1 with ammonia nitrogen as the only nitrogen source under aerobic culture and the changes of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen;

[0025] Figure 5 The growth of the strain Hunan Pseudomonas PAD-1 and the changes of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen under aerobic culture when nitrate nitrogen is the only nitrogen source;

[0026] Figure 6 The effect of different pyrite contents on denitrification of the strain Hunan Pseudomonas PAD-1 under aerobic culture, where a is total nitrogen, b is nitrate nitrogen, c is nitrite nitrogen, and d is ammonia nitrogen;

[0027] Figure 7 The enhancement effect of pyrite on denitrification of Pseudomonas hunanensis PAD-1 at different C / N under aerobic culture, where a is total nitrogen, b is nitrate nitrogen, c is nitrite nitrogen, and d is ammonia nitrogen;

[0028] Figure 8 The effect of pyrite on the respiration and nitrogen conversion rate of Hunan Pseudomonas PAD-1 under aerobic culture, where the left picture is the respiration and the right picture is the nitrogen conversion rate;

[0029] Fig. 9The effect of pyrite on the electron transport chain of Pseudomonas australis PAD-1 under aerobic culture, where a is Zeta potential, b is the content of NADH, c is the electrochemical impedance spectroscopy (EIS spectrum), d is the content of ETSA, Control is the control group (without pyrite addition), and Pyrite is the treatment group containing pyrite;

[0030] Fig.10 The effect of pyrite on the enzyme activity of Pseudomonas australis PAD-1 under aerobic culture, where a is the protein content, b is the activity of Complex I, c is the activity of Complex III, d is the activity of Cyt C, Control is the control group (without pyrite addition), Pyrite is the treatment group containing pyrite, and protien concentration is the protein concentration;

[0031] Fig.11 Diagram of the mechanism of pyrite-enhanced electron transfer.

[0032] Biological deposit information

[0033] Pseudomonas hunanensis strain PAD-1 was deposited in the China Center for Type Culture Collection on September 6, 2022. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO:M 20221381, and the deposit postal code is 430072. DETAILED DESCRIPTION

[0034] In the present invention, "C / N ratio" refers to the carbon-nitrogen ratio, where C refers to carbon, and N refers to nitrogen. The carbon-nitrogen ratio refers to the ratio of the total carbon content to the total nitrogen content in organic matter.

[0035] In the present invention, "culture" refers to a product prepared by inoculating bacteria into any suitable culture medium. The type of culture medium can be replaced according to different needs, including optimization of the components of commonly used culture media.

[0036] In the present invention, "preparation" refers to a product prepared by the strain of the present invention, and the preparation method thereof may be any method in the prior art or any possible method in the future. The characteristic of the preparation is that the preparation is carried out by the strain of the present invention, and the preparation method may be diverse.

[0037] In the present invention, "nitrogen-rich wastewater" refers to wastewater containing a relatively large amount of nitrogen, and the forms of nitrogen include but are not limited to nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen.

[0038] The present invention provides a Hunan Pseudomonas strain PAD-1 (Pseudomonas hunanensis strain PAD-1), and the Hunan Pseudomonas strain PAD-1 has a deposit number of CCTCC NO: M 20221381. The colony morphology of the Hunan Pseudomonas strain PAD-1 of the present invention is that the colonies are round, white, moist, and have a smooth surface. The bacteria are rod-shaped and are about 0.1-0.2×1-2.0 μm. The Hunan Pseudomonas strain PAD-1 provided by the present invention has the function of aerobic denitrification, can directly convert nitrate nitrogen into gas products, has less accumulation of nitrite nitrogen and ammonia nitrogen, and can effectively treat wastewater.

[0039] The growth performance of Hunan Pseudomonas strain PAD-1 is similar to that of aerobic denitrifying strains Methylobacterium gregans DC-1 and Pseudomonas sutzeri AD-1 isolated in the laboratory earlier. Hunan Pseudomonas strain PAD-1 has a higher removal rate. Within 60 hours, NO 3 - The -N content decreased from the initial value of 200 mg / L to 21.21 mg / L, and the removal rate reached 2.98 mg / L / h, which was higher than the removal rates of aerobic denitrification strains Methylobacterium gregans DC-1, Pseudomonasstutzeri AD-1 and the reported strains Rhodococcus sp.CPZ24, Pannonibacterphragmitetus B1 and Enterobacter cloacae HW-15. The removal rates of aerobic denitrification strains Methylobacterium gregans DC-1 were 2.73 mg / L / h, Pseudomonas stutzeriAD-1 was 2.08 mg / L / h, Rhodococcus sp.CPZ24 was only 0.93 mg / L / h, Pannonibacterphragmitetus B1 was 0.81 mg / L / h, and Enterobactercloacae The removal rate of HW-15 was 0.28 mg / L / h. Therefore, the above results show that Hunan Pseudomonas strain PAD-1 has a strong effect on NO 3 - -N has a high removal efficiency and has potential application prospects in bioremediation in wastewater treatment systems.

[0040] Among them, the aerobic denitrifying strain Methylobacterium gregans DC-1 was disclosed in "Oscar Omondi Donde, Bangding Xiao. Efficacy of zero nitrous oxide emitting aerobic denitrifying bacterium, Methylobacterium gregans DC-1 in nitrate removal with strong auto-aggregation property. Bioresource Technology" (Pei Hong, Yilin Shu, Xingqiang Wu, Chunbo Wang, Cuicui Tian, Hailong Wu, Oscar Omondi Donde, Bangding Xiao. Efficacy of zero nitrous oxide emitting aerobic denitrifying bacterium, Methylobacterium gregans DC-1 in nitrate removal with strong auto-aggregation property. Bioresource Technology, 2019, 293), and Pseudomonas stutzeri AD-1 was disclosed in "Novel heterotrophic nitrogen removal and assimilation characteristic of the newly isolated bacterium Pseudomonas stutzeri AD-1. Journal of Bioscience and Bioengineering" (Hui Qing, Oscar Omondi Donde, Cuicui Tian, Chunbo Wang, Xingqiang Wu, Shanshan Feng, Yao Liu, Bangding Xiao. Novel heterotrophic nitrogen removal and assimilation characteristic of the newly isolated bacterium Pseudomonas stutzeri AD-1.Journal of Bioscience and Bioengineering, 2018, 126: 339 - 345), Rhodococcus sp. CPZ24 was disclosed in "Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24. Bioresour. Technol." (P. Chen, J. Li, Q. X. Li, Y. Wang, S. Li, T. Ren, L. Wang. Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24. Bioresour. Technol., 2012, 116: 266 - 270), Pannonibacter phragmitetus B1 was disclosed in "High - efficiency inorganic nitrogen removal by newly isolated Pannonibacter phragmitetus B1. Bioresour. Technol." (H. Bai, S. Liao, A. Wang, J. Huang, W. Shu, J. Ye. High - efficiency inorganic nitrogen removal by newly isolated Pannonibacter phragmitetus B1. Bioresour. Technol., 2019, 271: 91 - 99), Enterobacter cloacae HW - 15 was disclosed in "Removal of nitrogen and phosphorus by Heterotrophic nitrification - aerobic denitrification of a denitrifying phosphorus - accumulating bacterium Enterobacter cloacae HW - 15." (W. J. Wan, D. L. He, Z. J. Xue.Removal of nitrogen and phosphorus by Heterotrophic nitrification-aerobic denitrification of a denitrifying phosphorus-accumulatingbacteriumEnterobacter cloacae HW-15.Ecol.Eng.,2014,99:199-208). .

[0041] The present invention also preferably provides a method for cultivating the Hunan Pseudomonas strain PAD-1 described in the above technical solution, comprising the following steps: inoculating the Hunan Pseudomonas strain PAD-1 in a culture medium to obtain a Hunan Pseudomonas strain PAD-1 seed solution; inoculating the Hunan Pseudomonas strain PAD-1 seed solution into a DBM culture medium for expansion culture to obtain an expanded Hunan Pseudomonas strain PAD-1. The culture medium of the present invention preferably includes but is not limited to: denitrification enrichment medium (DBM for short), bromothymol blue medium (BTB for short) and aerobic denitrification test medium (ADM for short); the inoculation amount of the Hunan Pseudomonas strain PAD-1 is preferably 1% to 5% of the volume of the culture medium, more preferably 2 to 4%; in a specific embodiment of the present invention, the inoculation amount of the southern Pseudomonas strain PAD-1 is preferably 1% or 5% of the volume of the culture medium; the culture time is preferably 2.5d to 7d, and is also preferably 3 to 6d, more preferably 4 to 5d, the temperature is 25 to 30°C, and the speed is 200r / min. The inoculation amount of the Hunan Pseudomonas strain PAD-1 seed solution of the present invention is preferably 1% of the volume of the DBM culture medium; the time of the expanded culture is preferably 5d to 7d, the temperature is preferably 25 to 30°C, further preferably 26 to 29°C, and more preferably 27 to 28°C; the components and dosage of the DBM are preferably as shown in Table 1, and DBM is also called denitrification enrichment medium.

[0042] Table 1 Composition of denitrification enrichment medium

[0043]

[0044]

[0045] Note: Each liter of trace element solution contains 1.80 g FeCl 2 ·4H 2 O, 0.70 gMnCl 2 ·4H 2 O, 0.50g ZnCl 2 , 0.50gH 3 BO 3 , 0.25gCoCl2 6H 2 O, 0.03gNa 2 MoO 4 ·2H 2 O, 0.01gNiCl 2 6H 2 O, 0.01gNa 2 SeO 3 ·5H 2 O and 0.01 g CuCl 2 ·2H 2 O.

[0046] The present invention provides a wastewater treatment agent, wherein the effective ingredients of the wastewater treatment agent include the Hunan Pseudomonas strain PAD-1 and / or the Hunan Pseudomonas strain PAD-1 preparation described in the above technical solution. 600 Preferably it is 0.91 to 1.63, more preferably 1 to 1.63, more preferably 1.5 to 1.63, and most preferably 1.63. The Hunan Pseudomonas strain PAD-1 preparation of the present invention preferably includes one or more of the Hunan Pseudomonas strain PAD-1 culture, culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant and fermentation broth extract, and more preferably includes the Hunan Pseudomonas strain PAD-1 lyophilized powder or the Hunan Pseudomonas strain PAD-1 fermentation broth. The preparation method of the Hunan Pseudomonas strain PAD-1 culture, culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant and fermentation broth extract of the present invention is not particularly limited, and can be prepared in a manner well known to those skilled in the art.

[0047] The effective component of the wastewater treatment agent of the present invention also preferably includes pyrite, more preferably pyrite. The concentration of pyrite in the wastewater treatment agent of the present invention is preferably 0.8-3.2 g / L, more preferably 0.8-1.6 g / L, and most preferably 1.6 g / L.

[0048] The present invention provides the use of the Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution in wastewater treatment. Further, the present invention provides the use of the Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution in the treatment of nitrogen-rich wastewater. The Hunan Pseudomonas strain PAD-1 or the wastewater treatment agent described in the present invention can treat nitrogen-rich wastewater with a nitrate nitrogen concentration of 10 to 200 mg / L, more preferably 200 mg / L, a nitrite nitrogen concentration of 10 to 200 mg / L, an ammonia nitrogen concentration of preferably 10 to 200 mg / L, and a total nitrogen concentration of preferably 10 to 200 mg / L; the pH value of the nitrogen-rich wastewater is preferably 5 to 9, more preferably 6 to 8.

[0049] The present invention provides a method for treating wastewater, the method comprising the following steps:

[0050] The Hunan Pseudomonas strain PAD-1 described in the above technical solution or the wastewater treatment agent described in the above technical solution is mixed with wastewater to treat the wastewater.

[0051] The inoculation amount of the Hunan Pseudomonas strain PAD-1 of the present invention is preferably 1% to 5% of the volume of the wastewater, more preferably 2 to 4%; the volume of the wastewater treatment agent is preferably 1% to 5% of the volume of the wastewater, more preferably 2 to 4%; in a specific embodiment of the present invention, the inoculation amount of the Hunan Pseudomonas strain PAD-1 is preferably 1% or 5% of the volume of the wastewater. The wastewater treatment method of the present invention is preferably aerobic culture, also known as natural oxygen-enriched culture; the aerobic culture time is preferably 2.5d to 7d, and the temperature is 25 to 30°C.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Medium preparation:

[0054] The composition of aerobic denitrification test medium (ADM) is as follows: 1.44 g / L KNO 3 , 1.50g / LKH 2 PO 4 , 16.67g / LC 4 H 4 Na 2 O 4 6H 2 O, 5.27 g / LNa 2 HPO 4 12H 2 O, 0.10g / LMgSO 4 , 2mL trace element solution; each liter of trace element solution contains 1.80g FeCl 2 ·4H 2 O, 0.70 g MnCl 2 ·4H 2 O, 0.50g ZnCl 2 , 0.50g H 3 BO 3 , 0.25gCoCl 2 6H 2 O, 0.03 g Na 2 MoO 4 ·2H 2 O, 0.01 g NiCl2 6H 2 O, 0.01 g Na 2 SeO 3 ·5H 2 O and 0.01 g CuCl 2 ·2H 2 O.

[0055] Bromothymol blue medium (BTB) is as follows: C 6 H 5 Na 3 O 7 8.50 g / L, KNO 3 1.00 g / L, KH 2 PO 4 1.00g / L, FeSO 4 7H 2 O 0.05g / L, CaCl 2 0.20 g / L, MgSO 4 7H 2 O 1.00 g / L, 1% bromothymol blue 1 mL and Agar 20 g / L.

[0056] Example 1 Screening and Isolation of Bacteria

[0057] Sample collection: taken from Dianchi Lake sediments.

[0058] Enrichment culture and isolation of strains: Take 5g of sediment and add it to 100mL of denitrification enrichment medium (DBM), and culture it in a shaker at 30℃ and 120r / min for 1 week. Take 10mL of enrichment solution and spread it on a plate made of bromothymol blue medium (BTB), and invert it in a 30℃ constant temperature incubator for culture. After single colonies grow on the solid plate, pick different single colonies for separation, purification and numbering.

[0059] Strain screening: The isolated and purified strains were inoculated into the denitrification test medium (ADM) at 5% of the volume of the denitrification test medium (ADM) and cultured at 30°C and 200r / min. After 24h, the total nitrogen (TN) concentration in the culture medium was determined. Finally, 20 strains with high-efficiency denitrification ability (total nitrogen rate within 24 hours was higher than 95%) were screened, and it was found through visual observation that the extracellular secretion of one new strain was significantly higher than that of other bacteria. This strain can form large flocs with pyrite, so this genus was selected as the object of subsequent experimental research and recorded as strain PAD-1.

[0060] Example 2 Morphological and molecular biological identification of strain PAD-1

[0061] The colony morphology and cell morphology of strain PAD-1 were observed by scanning electron microscopy. Figure 1 It can be seen that the colony morphology of strain PAD-1 is as follows: the colonies are round, white, moist, with a smooth surface, and the bacteria are rod-shaped, about 0.1-0.2×1-2.0 μm.

[0062] The strain PAD-1 was inoculated into aerobic denitrification test medium (ADM) at an inoculation amount of 5% of the volume of the nitrogen-rich wastewater medium, and cultured at 30°C with constant temperature shaking at 200 r / min for 100 h to obtain a seed solution.

[0063] Figure 2 As shown, Figure 2 The Strain PAD-1 in the reference is strain PAD-1.

[0064] Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification characteristics of strain PAD-1, strain PAD-1 was identified as Hunan Pseudomonas and named Hunan Pseudomonas PAD-1.

[0065] Example 3 Cultivation method of Hunan Pseudomonas PAD-1

[0066] Hunan Pseudomonas PAD-1 was inoculated into aerobic denitrification test medium (ADM) at an inoculation amount of 5% of the volume of nitrogen-rich wastewater medium, and cultured at 30°C with constant temperature shaking at 200 r / min for 100 h to obtain seed solution;

[0067] The seed solution was inoculated into DBM medium for expansion culture. The expansion culture time was 7 days, the temperature was 30° C., and the inoculation amount was 1% of the volume of the DBM medium to obtain Hunan Pseudomonas PAD-1 bacterial solution.

[0068] Example 4 Amplification of nitrogen pathway-related genes of Hunan Pseudomonas PAD-1

[0069] The Hunan Pseudomonas PAD-1 bacterial solution obtained by culture in Example 3 was used as a template to amplify nitrogen pathway related genes in Hunan Pseudomonas PAD-1. The related genes are: NapA, NarG, NirS, NorB and NosZ genes. The amplification primers are shown in Table 2.

[0070] Table 2 Detailed information of amplification primers

[0071]

[0072]

[0073] Note: NapA is the periplasmic nitrate reductase gene, NarG is the nitrate reductase gene, NirS is the nitrite reductase gene, NorB is the nitric oxide reductase gene, and NosZ is the nitrous oxide reductase gene.

[0074] The amplification system was a 25 μL system, which included: 12.5 μL of 2xES Taq MasterMix, 1.0 μL of upstream primer and downstream primer, 0.5 μL of template, and 10 μL of double-distilled water;

[0075] The amplification conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of: 94°C for 30 s; annealing for 20 s, with the annealing temperatures of NapA at 62°C, NarG at 57°C, NirS at 57°C, NorB at 58°C, and NosZ at 57°C; and extension at 72°C for 5 min.

[0076] Amplification results are shown in Figure 3 , where M is a marker. The results showed that Hunan Pseudomonas PAD-1 presented a relatively complete denitrification pathway, and the periplasmic nitrate reductase gene, nitrate reductase gene, nitrite reductase gene and nitrous oxide reductase gene were successfully amplified.

[0077] Example 5

[0078] Hunan Pseudomonas PAD-1 was inoculated into 500 mL of a culture medium with ammonia nitrogen as the sole nitrogen source for aerobic culture. The inoculation amount was 1% of the volume of the culture medium with ammonia nitrogen as the sole nitrogen source. The composition of the culture medium with ammonia nitrogen as the sole nitrogen source was 0.76 g / LNH 4 + -N, 1.50g / LKH 2 PO 4 , 16.67g / LC 4 H 4 Na 2 O 4 6H 2 O, 5.27 g / LNa 2 HPO 4 12H 2 O, 0.10 g / L MgSO 4 , 2mL of trace elements; each liter of trace elements contains 1.80g FeCl 2 ·4H 2 O, 0.70 g MnCl 2 ·4H 2 O, 0.50g ZnCl 2 , 0.50g H 3 BO 3 , 0.25g CoCl 2 6H 2 O, 0.03 g Na 2 MoO 4 2H2O, 0.01g NiCl 2 6H 2 O, 0.01gNa 2 SeO 3 ·5H 2 O and 0.01 g CuCl 2 ·2H2 O, the concentration of ammonia nitrogen was 200 mg / L, the aerobic culture time was 120 h, and the temperature was 30 °C.

[0079] Example 6

[0080] Hunan Pseudomonas PAD-1 was inoculated into 500 mL of culture medium with nitrate as the sole nitrogen source for aerobic culture. The inoculation amount was 1% of the volume of the culture medium with nitrate as the sole nitrogen source. The composition of the culture medium with nitrate as the sole nitrogen source was 1.44 g / L KNO 3 , 1.50g / LKH 2 PO 4 , 16.67g / LC 4 H 4 Na 2 O 4 6H 2 O, 5.27 g / LNa 2 HPO 4 12H 2 O, 0.10g / LMgSO 4 , 2mL of trace elements; each liter of trace elements contains 1.80g FeCl 2 ·4H 2 O, 0.70 g MnCl 2 ·4H 2 O, 0.50g ZnCl 2 , 0.50g H 3 BO 3 , 0.25g CoCl 2 6H2O, 0.03g Na 2 MoO 4 ·2H 2 O, 0.01 g NiCl 2 6H 2 O, 0.01gNa 2 SeO 3 ·5H 2 O and 0.01 g CuCl 2 ·2H 2 O, the concentration of nitrate nitrogen was 200 mg / L, the aerobic culture time was 120 h, and the temperature was 30 °C.

[0081] In Example 5 and Example 6, the effects of different nitrogen sources on ammonia nitrogen, nitrate nitrogen, nitrite nitrogen in wastewater and the OD of Hunan Pseudomonas PAD-1 in wastewater were measured at 0, 3h, 6h, 9h, 12h, 18h, 24h, 30h, 36h, 48h, 60h, 90h and 120h of aerobic culture. 600 and LogoOD 600The influence of nitrate nitrogen and nitrite nitrogen was determined by ion chromatography, and ammonia nitrogen was determined by Nessler's reagent spectrophotometry. The results of Example 5 are shown in Figure 4 The results of Example 6 are shown in Figure 5 ,Depend on Figure 4 and Figure 5 It can be seen that under aerobic culture, strain PAD-1 can grow stably and achieve stable denitrification when ammonia nitrogen and nitric nitrogen are the only nitrogen sources.

[0082] Example 7 Effect of pyrite content on denitrification by Pseudomonas hunanensis PAD-1

[0083] Hunan Pseudomonas PAD-1 was cultured in a denitrification enrichment medium at 30°C to the logarithmic phase, and the Hunan Pseudomonas PAD-1 in the logarithmic phase was inoculated into a nitrogen-rich wastewater medium containing pyrite. The inoculation amount was 1% of the volume of the nitrogen-rich wastewater medium. The temperature was 30°C, the shaker speed was 200r / min, and the aerobic culture was carried out for 100h. The concentrations of pyrite in the nitrogen-rich wastewater medium containing pyrite were 0.8g / L, 1.6g / L and 3.2g / L, respectively. The C / N in the denitrification enrichment medium was 15. The nitrogen-rich wastewater medium was used as the blank control group, recorded as Control.

[0084] The effects of different amounts of pyrite on total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in nitrogen-rich wastewater were determined. Nitrate nitrogen and nitrite nitrogen were determined by ion chromatography, ammonia nitrogen was determined by Nessler's reagent spectrophotometry, and total nitrogen was the sum of various forms of nitrogen. Figure 6 As shown in the results, it was found that the addition of pyrite could significantly enhance the denitrification process of Hunan Pseudomonas PAD-1. When the pyrite concentration was 0.8-1.6 g / L, the soluble total nitrogen removal rate was higher than 97% at 18 h, which was much higher than the 6% of the blank control group in the same period. However, the denitrification enhancement effect would decrease if the pyrite concentration was continuously increased. Figure 6 The upper left line graph shows that when the pyrite concentration is 3.2 g / L, the TN removal rate of PAD-1 is only 18.6% at 18 h, but it can reach 97.1% at 24 h, which is much higher than the 28.5% of the control group. Therefore, the optimal addition amount of pyrite is 1.6 g / L.

[0085] Example 8 Denitrification of strain PAD-1 at different C / N ratios

[0086] Hunan Pseudomonas PAD-1 was inoculated into denitrification enrichment medium with C / N ratios of 0, 2:1, 5:1, 10:1 and 15:1, respectively. The medium used in this part was aerobic denitrification test medium (ADM), and the C in the medium was adjusted as needed. 4 H 4 Na 2 O 4 6H2 The C / N ratio was adjusted by adjusting the concentration of O, the concentration of pyrite was 1.6 g / L, the inoculation amount was 1% of the volume of the aerobic denitrification test medium, the temperature was 30°C, the shaker speed was 200 r / min, and the aerobic culture was carried out for 60 h, which was recorded as the treatment group containing pyrite, i.e., Pyrite. The aerobic denitrification test medium (ADM) without pyrite and with a C / N ratio of 0, 2:1, 5:1, 10:1 and 15:1 was used as the control group, i.e., Control.

[0087] The effects of different amounts of pyrite on total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in nitrogen-rich wastewater were determined. Nitrate nitrogen and nitrite nitrogen were determined by ion chromatography, ammonia nitrogen was determined by Nessler's reagent spectrophotometry, and total nitrogen was the sum of various forms of nitrogen. Figure 7 As shown, the total nitrogen removal rate of the treatments containing pyrite was improved compared with the control group. In the range of C / N=2-15:1, the enhancing effect of pyrite on PAD-1 denitrification was positively correlated with C / N. In addition, the best enhancing effect was achieved at C / N=15:1. In addition, at C / N=5:1 and C / N=10:1, the total nitrogen removal rate of the pyrite group reached more than 90% at 60h, which was better than the control group. In addition, as Figure 7 As shown in Figure c, pyrite significantly reduced nitrite accumulation at 18 h and enhanced denitrification. At low C / N, especially at C / N = 2:1, a small amount of ammonia nitrogen accumulated in the pyrite group. Therefore, at very low C / N, nitrate can be converted into ammonia nitrogen together with pyrite and denitrifiers.

[0088] The effects of pyrite on the respiration and nitrogen conversion rate of Pseudomonas hunanensis PAD-1 were determined by GC. The CO in the pyrite-containing group and the control group at 60 h was measured. 2 and N 2 The amount of nitrogen generated was used for the respiration and nitrogen conversion rate of each group. The experimental results are shown in Table 2 and Figure 8 .

[0089] Table 2 Respiration and nitrogen conversion rate of different treatments

[0090]

[0091] The experimental results are shown in Table 2 and Figure 8 As shown, the respiration intensity of Pseudomonas hunanensis PAD-1 was positively correlated with C / N. After in-depth analysis, the respiration rate of Pseudomonas hunanensis PAD-1 in the pyrite-containing treatment increased significantly compared with the control group, indicating that pyrite can increase the activity of Pseudomonas hunanensis PAD-1 and accelerate its cell metabolism rate. Therefore, a higher cell metabolism rate will promote the decomposition of organic carbon and further provide more electron donors for denitrification. Figure 8As shown in b, after 60 hours of cultivation, the nitrogen conversion rate of Hunan Pseudomonas PAD-1 reached 38.34%, an increase of 7.62%.

[0092] Example 9 Effects of pyrite on the electron transport chain, protein and enzyme activity of Hunan Pseudomonas PAD-1

[0093] Hunan Pseudomonas PAD-1 was inoculated into an aerobic denitrification test medium (ADM) culture medium with a C / N ratio of 15:1, and the concentration of pyrite in the aerobic denitrification test medium was 1.6 g / L. The inoculation amount was 1% of the volume of the aerobic denitrification test medium. The culture was carried out at a temperature of 30°C, a shaking speed of 200 r / min, and aerobic culture for 60 hours. The treatment group containing pyrite was used as the control group, and the aerobic denitrification test medium without pyrite added was used as the control group.

[0094] The effects of pyrite on the electron transport chain, protein and enzyme activity of Hunan Pseudomonas PAD-1 were determined. The detection method of the electron transport chain was based on the reference Zhang Y, Lu C, Chen Z, et al. Multifaceted synergistic electron transfer mechanism for enhancing denitrification by clay minerals [J]. Science of the Total Environment, 2022, 812. The enzyme activity was detected by ELISA kit, the protein was detected by Coomassie Brilliant Blue, and the electron transport chain detection results were shown in Fig. 9 , Table 3 and Table 4, protein and enzyme activity test results are shown in Fig.10 and Table 5, Fig.11 This is only a flow chart of electron transfer for the present invention.

[0095] Table 3 Detection results of electron transport chain

[0096]

[0097] Table 4 Data of electrochemical impedance spectroscopy

[0098]

[0099]

[0100]

[0101] Table 5 Detection results of protein and enzyme activity

[0102]

[0103] The results of the electron transport chain test are Fig. 9 , Tables 3 and 4 show that the addition of pyrite significantly increased the NADH abundance of Pseudomonas hunanensis PAD-1, which provided more electrons for the electron transport system (ETS) and helped to accelerate the electron transport system activity (ETSA). Fig. 9 Figure c is an electrochemical impedance spectroscopy (EIS), which shows that pyrite promotes electron transfer. The actual ETSA of the control group and the pyrite group showed that after the addition of pyrite, the electron transfer of strain PAD-1 increased by 1.8 times. Therefore, pyrite strengthened the electron transfer system of strain PAD-1.

[0104] Protein and enzyme activity assays were performed by Fig.10 From Table 5, we can see that Fig.10 The a in Figure 2 shows that the addition of pyrite significantly increased the protein content by 3.07 times, which may help increase the number of specific denitrification enzymes to promote accelerated denitrification. In the electron transport chain, NADH generates electrons through the action of Complex I, and then the electrons are transferred to Complex III and finally to the cell cycle protein Cyt C in the periplasm. Fig.10 As can be seen from b and c in the figure, the addition of pyrite has no significant effect on Complex I, but significantly increases the activity of Complex III by 28%, which promotes the formation of more electrons in the electron transport system. Cyt C is an important electron transporter in the electron transport chain, responsible for transferring electrons from Complex III to denitrification enzymes. Fig.10 As can be seen in Figure d, the addition of pyrite does not affect the activity of Cyt C. Therefore, Cyt C is not the main reason for the increase in the activity of the electron transport system.

[0105] In summary, the Hunan Pseudomonas strain PAD-1 of the present invention has aerobic denitrification ability, can directly convert nitrate nitrogen into gas products, and has less accumulation of nitrite nitrogen and ammonia nitrogen, further achieving the effect of effectively treating low C / N wastewater, while also having the advantages of low cost and green environmental protection. The strain shows excellent performance in treating low-carbon wastewater, and its cultivation method is simple to operate, with low energy consumption and cost. Moreover, the strain of the present invention can be coupled with pyrite to treat low C / N wastewater, which is easy to use, simple to operate, and reduces the cost of sewage treatment. The strain can be coupled with pyrite to post-treat low C / N wastewater, and the treatment effect is more significant.

[0106] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Pseudomonas hunanensis strain PAD-1, characterized in that: The deposit number of the Hunan Pseudomonas strain PAD-1 is CCTCC NO:M 20221381.

2. A wastewater treatment agent, characterized in that The effective ingredients of the wastewater treatment agent include the Hunan Pseudomonas strain PAD-1 and / or the Hunan Pseudomonas strain PAD-1 preparation according to claim 1; The Hunan Pseudomonas strain PAD-1 preparation is one or more of the Hunan Pseudomonas strain PAD-1 culture, lyophilized powder and fermentation liquid.

3. The wastewater treatment agent according to claim 2, characterized in that The OD600 of the Hunan Pseudomonas strain PAD-1 in the wastewater treatment agent is 0.91-1.

63.

4. The wastewater treatment agent according to claim 2, characterized in that The effective component of the wastewater treatment agent also includes pyrite.

5. The wastewater treatment agent according to claim 4, characterized in that The concentration of pyrite in the wastewater treatment agent is 0.8-3.2 g / L.

6. Use of the Hunan Pseudomonas strain PAD-1 described in claim 1 or the wastewater treatment agent described in any one of claims 2 to 5 in the treatment of low C / N wastewater and / or nitrogen-rich wastewater.

7. A method for treating low C / N wastewater and / or nitrogen-rich wastewater, characterized in that: The processing method comprises the following steps: The Hunan Pseudomonas strain PAD-1 described in claim 1 or the wastewater treatment agent described in any one of claims 2 to 5 is mixed with low C / N wastewater and / or nitrogen-rich wastewater to perform wastewater treatment.

8. The processing method according to claim 7, characterized in that: The inoculation amount of the Hunan Pseudomonas strain PAD-1 is 1% to 5% of the volume of the low C / N wastewater and / or nitrogen-rich wastewater.

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