Microbiological treatment method for green and efficient removal of nitrate nitrogen in high-salinity wastewater

Abstract

The invention belongs to the technical field of nitrate nitrogen wastewater treatment, and particularly relates to a microbiological treatment method for green and efficient removal of nitrate nitrogen in high-salinity wastewater. According to the method, the nitrate nitrogen in the wastewater is removed by inoculating a methylophilus with a nitrate assimilation reduction function into the wastewater containing the nitrate nitrogen, the nitrate nitrogen in the final effluent reaches the standard, intermediate products such as nitrous oxide and nitrogen are not generated, and meanwhile, the nitrogen removed from the wastewater can be recycled by collecting biomass precipitates. Compared with a traditional biological denitrification process, the method has the advantages that nitrate nitrogen and organic matters can be removed in a single reactor, and the requirement on the scale of the reactor and the subsequent energy cost are reduced; meanwhile, the microbiological treatment method disclosed by the invention is simple to operate, mild in reaction, high in nitrate nitrogen removal efficiency and applicable to denitrification treatment in wastewater with a wide salinity range, and has a wide application value in sewage treatment engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of nitrate nitrogen wastewater treatment, and more specifically, relates to a green and efficient microbial treatment method for removing nitrate nitrogen in high-salinity wastewater. Background Art

[0002] Nitrogen (N) is an important chemical element. The cycle of nitrogen in the ecosystem is crucial to the ecological balance. With the frequent industrial production activities and the increase in the application of agricultural nitrogen fertilizers, a large amount of active nitrogen exists in the wastewater generated by human activities. Direct discharge will cause a huge nitrogen load on the natural ecological environment, thus triggering a series of ecological problems caused by nitrogen pollution. At present, the main treatment approach for nitrate nitrogen in wastewater is biological denitrification. Through the nitrification-denitrification process, ammonia nitrogen is first oxidized to nitrate nitrogen (nitrification), and then nitrate nitrogen is reduced to nitrogen gas (denitrification) to effectively remove nitrate nitrogen in water. However, during the nitrification-denitrification process, N2O may be released as a by-product, which as a potent greenhouse gas exacerbates global warming. On the other hand, it is challenging to remove nitrate nitrogen from high-salinity wastewater. High-salinity environment inhibits the metabolism and growth of microorganisms. The growth rate of many microorganisms will be significantly reduced. Salt will inhibit enzyme activity and cell metabolic processes, resulting in a slowdown in the rate of cell proliferation, thereby affecting the efficiency of nitrogen removal by microorganisms.

[0003] Microorganisms with aerobic heterotrophic reduction of nitrate nitrogen have been studied, in which the assimilation and absorption process of nitrate nitrogen by microorganisms is one of the important processes of nitrogen conversion in the environment. After nitrate nitrogen is assimilated and reduced to ammonium by heterotrophic bacteria, the synthesized biomass nitrogen is stored in the microorganism, and no intermediate products such as N2O are produced. It is an environmentally friendly nitrogen conversion process. Compared with the traditional biological denitrification process performed by autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria, nitrate assimilating bacteria can remove nitrate nitrogen and organic matter in a single reactor, reducing the requirements for reactor scale and subsequent energy costs. At the same time, the path of removing nitrate nitrogen through the assimilation process is shorter, which means that microorganisms may be able to remove nitrogen in the environment faster than nitrification-denitrification processes. For example, Chinese patent application CN103923867A discloses a mixed bacterial microbial preparation composed of Bacillus, Pseudomonas stutzeri, Pseudomonas pseudoalcaligenes and Pseudomonas oleovorans, which is used to treat nitrate nitrogen-containing wastewater. It takes 4 days of culture to achieve complete denitrification, and the denitrification efficiency still needs to be further improved.

[0004] Therefore, there is an urgent need to construct a green and environmentally friendly method to remove nitrate nitrogen from high-salinity wastewater to meet the challenges of high salinity and high reactive nitrogen content in current human activity wastewater. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art that microorganisms are required to treat nitrate-containing wastewater for 4 days before complete denitrification can be achieved, and the denitrification efficiency still needs to be further improved, and provide the application of methylophaga murata in treating high nitrate-nitrogen wastewater.

[0006] Another object of the present invention is to provide a green and efficient microbial treatment method for removing nitrate nitrogen from high-salinity wastewater.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The invention protects the use of methylophaga in treating high nitrate nitrogen wastewater. The methylophaga is Methylophaga murata, which has been preserved in Guangdong Microbiological Culture Collection Center on February 28, 2012, with a preservation number of GDMCC NO: 1.508.

[0009] Furthermore, the above-mentioned Methylophaga murata was later renamed as Methylophaga muralis.

[0010] The present invention provides a microbial treatment method for removing nitrate nitrogen from high-salinity wastewater, comprising the following steps: adjusting the pH value of the nitrate nitrogen-containing wastewater to 6-9, inoculating Methylophaga murata bacterial solution therein, fully mixing, and performing microbial denitrification under aerobic conditions;

[0011] Wherein, the Methylophaga murata bacterial liquid is added into the nitrate-containing wastewater in the form of being attached to a microbial carrier or directly;

[0012] The Methylophaga murata was deposited in Guangdong Microbiological Culture Collection Center on February 28, 2012, with the deposit number of GDMCC NO: 1.508.

[0013] Furthermore, the OD of the Methylophaga murata bacterial solution is 600 Value ≥ 0.8.

[0014] Preferably, the inoculation amount of the Methylophaga murata bacterial solution is 5% to 40% by volume of the nitrate-containing wastewater.

[0015] Furthermore, the Methylophaga murata bacterial liquid is added into the nitrate-containing wastewater in the form of being attached to a microbial carrier or directly.

[0016] Furthermore, when the Methylophaga murata bacterial solution is directly added into the nitrate-nitrogen-containing wastewater, the inoculation amount of the Methylophaga murata bacterial solution is 5% to 15%, more preferably 8 to 12%.

[0017] Furthermore, when the Methylophaga murata bacterial liquid is added into the nitrate-nitrogen-containing wastewater in the form of being attached to a microbial carrier, the inoculation amount of the Methylophaga murata bacterial liquid is 30% to 40%.

[0018] When the scale of wastewater treatment is small and the volume of wastewater containing nitrate nitrogen is ≤1L, the Methylophaga murata bacterial solution can be directly added to the wastewater containing nitrate nitrogen to quickly carry out the microbial denitrification process.

[0019] When the scale of wastewater treatment is large and the volume of wastewater containing nitrate nitrogen is greater than 1L, the Methylophaga murata bacterial solution is added to the wastewater containing nitrate nitrogen in the form of attachment to a microbial carrier (i.e., immobilized microbial technology), which can more quickly carry out the microbial denitrification process. This is because the immobilized microbial technology can make the microorganisms highly dense and maintain biological activity, and can multiply rapidly and in large quantities under suitable conditions. This technology is applied to wastewater treatment, which is conducive to increasing the concentration of microorganisms (especially microorganisms with special functions) in the bioreactor, helping the microorganisms resist the influence of adverse environments, and is conducive to solid-liquid separation after the reaction, shortening the time required for treatment.

[0020] Furthermore, the preparation method of the Methylophaga murata bacterial liquid comprises the following steps:

[0021] S1. Inoculate Methylophaga murata on solid culture medium and culture until a single colony grows;

[0022] S2. Pick up the single colony obtained in step S1 for seed culture to obtain seed solution;

[0023] S3. The seed solution obtained in step S2 is inoculated into a liquid culture medium for expansion culture to obtain a Methylophagamurata bacterial solution.

[0024] Furthermore, the OD 600 The value is the absorbance of the bacterial solution measured at a wavelength of 600 nm.

[0025] When the Methylophaga murata bacterial liquid is added to the nitrate-containing wastewater in the form of being attached to a microbial carrier, the preparation method of the Methylophaga murata bacterial liquid comprises the following steps:

[0026] S1. Inoculate Methylophaga murata on solid culture medium and culture until a single colony grows;

[0027] S2. Pick up the single colony obtained in step S1 for seed culture to obtain seed solution;

[0028] S3. Inoculate the seed solution obtained in step S2 into the liquid culture medium for expansion culture; when the OD of the bacterial solution 600 When the value is ≥0.8, a sterilized microbial carrier is added to the bacterial solution, and the culture is continued so that the Methylophaga murata bacterial solution is fully attached to the microbial carrier to obtain the Methylophaga murata bacterial solution.

[0029] Furthermore, the continued culture time is 10 to 14 hours.

[0030] Furthermore, the preferred sterilization condition is high pressure steam sterilization at 121° C. for 20 minutes.

[0031] Specifically, in step S3, the seed solution obtained in step S2 is inoculated into a liquid culture medium for expansion culture; a spectrophotometer is used to detect the absorbance of the bacterial solution at 600 nm to monitor the growth of the Methylophaga murata bacterial solution; 600 After the value is ≥0.8, sponge fillers and sand and gravel particles that have been sterilized with high-pressure steam at 121°C for 20 minutes and cooled naturally are added into the bacterial solution, the mass ratio of sponge fillers to sand and gravel particles is 1:2, and the bacterial solution is continued to be cultured for 12 hours at a temperature of 30°C and a rotation speed of 160 rpm to obtain Methylophaga murata bacterial solution.

[0032] Preferably, the microbial carrier includes one or more of sponge filler, sand and gravel particles, and activated carbon.

[0033] Furthermore, the Methylophaga murata bacterial liquid is fixed on the microbial carrier by physical adsorption.

[0034] Furthermore, the mixing ratio of the Methylophaga murata bacterial solution and the microbial carrier is 1: (15-30) L / g, preferably 1: 18 L / g.

[0035] Furthermore, the nitrate-nitrogen-containing wastewater includes nutrients.

[0036] Furthermore, the nutrients or liquid culture medium include carbon sources, nitrogen sources, inorganic salts, trace elements and vitamins.

[0037] Furthermore, the carbon source mainly comes from methanol. The carbon source provides energy and carbon skeleton required for cell growth.

[0038] Furthermore, the nitrogen source mainly comes from one or more of nitrate, ammonium salt and amino acid. The nitrogen source can provide nitrogen elements required for cells to synthesize proteins and nucleic acids.

[0039] Furthermore, the inorganic salt is mainly derived from one or more of phosphate, sulfate, magnesium salt, potassium salt, and sodium salt, and specifically includes one or more of potassium nitrate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, sodium bicarbonate, and sodium carbonate. Inorganic salts can provide the macroelements required for cell growth.

[0040] Further, the trace elements include one or more of iron, manganese, copper, zinc, cobalt, boron, and calcium. Specifically, iron can be derived from ferrous sulfate, manganese can be provided by manganese chloride tetrahydrate, boron can be provided by boric acid, calcium can be derived from calcium chloride, cobalt can be provided by cobalt chloride hexahydrate, copper can be provided by copper sulfate pentahydrate, and zinc can be provided by zinc sulfate heptahydrate. Although trace elements are required in small quantities, they are essential for cell growth.

[0041] Furthermore, the vitamin is preferably vitamin B12. Vitamins participate in various biochemical reactions within cells and promote cell growth.

[0042] Specifically, as a preferred embodiment, the nutrient substance (or liquid culture medium) (1L) contains potassium nitrate (1g), potassium dihydrogen phosphate (1g), magnesium sulfate (0.22g), sodium chloride (30g), methanol (10mL), trace element solution (1mL), vitamin B12 (20μg), and 2mol / L sodium bicarbonate solution and 1mol / L sodium carbonate solution are additionally prepared, and 50mL and 10mL are added to each liter of culture medium, respectively.

[0043] Furthermore, the trace element solution (1 L) comprises manganese chloride tetrahydrate (4.0 g), ferrous sulfate heptahydrate (2.0 g), boric acid (1.0 g), calcium chloride (1.0 g), cobalt chloride hexahydrate (1.6 g), copper sulfate pentahydrate (1.5 g), and zinc sulfate heptahydrate (3.0 g).

[0044] Preferably, the temperature of the microbial denitrification is 20-32°C, more preferably 28-32°C. The temperature of microbial denitrification is consistent with the growth temperature, because the microorganisms need to maintain good biological activity during the denitrification process. Methylophagamurata bacterial liquid can grow at the above temperature, maintain good activity, and perform microbial denitrification.

[0045] Furthermore, the culture temperature of the Methylophaga murata bacterial liquid is 20-32°C.

[0046] Preferably, the temperature of the Methylophaga murata bacterial liquid culture is 28-32°C.

[0047] Furthermore, the rotation speed of the Methylophaga murata bacterial liquid culture is 120 to 200 rpm, preferably 150 to 180 rpm.

[0048] Furthermore, the culture medium is sterilized before use, and the sterilization conditions are preferably: sterilization at 121° C. for 20 min, and cooling to room temperature before use.

[0049] Furthermore, after the culture medium is sterilized and cooled, the vitamin B12 solution is injected into the culture medium after passing through a 0.22 μm filter membrane.

[0050] Furthermore, the solid culture medium is a liquid culture medium with 1.5% to 3% (mass percentage of the liquid culture medium) of agar powder added thereto, preferably 2%.

[0051] Furthermore, the nitrate nitrogen in the nitrate nitrogen-containing wastewater is 10 to 200 mg / L. When the nitrate nitrogen concentration in the water body does not exceed 200 mg / L, the microbial treatment method of the present invention converts all the nitrate nitrogen contained in the culture medium into biomass nitrogen stored in the microorganism within 14 hours, thereby achieving the purpose of denitrification of the water environment.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The microbial treatment method of the present invention removes nitrate nitrogen in the wastewater by inoculating a salt-tolerant bacterium with nitrate assimilation and reduction function into the wastewater containing nitrate nitrogen. When the final effluent nitrate nitrogen reaches the standard, all nitrate nitrogen is converted into biomass nitrogen and stored in the microorganisms, and no intermediate products such as nitrous oxide and nitrogen gas are produced. At the same time, the nitrogen removed from the wastewater can be recovered by collecting biomass precipitates. Compared with the traditional biological denitrification process, this method can achieve the removal of nitrate nitrogen and organic matter in a single reactor, reducing the requirements for the reactor scale and subsequent energy costs; at the same time, the microbial treatment method of the present invention is simple to operate, mild in reaction, and has a high removal efficiency for nitrate nitrogen. It can be applied to denitrification treatment in wastewater with a wide range of salinity, and has broad application value in sewage treatment projects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a statistical chart of the denitrification effect of Methylophaga murata on a small scale in the laboratory.

[0055] Figure 2 Schematic diagram of the reactor.

[0056] Figure 3 This is a statistical graph of the growth of Methylophaga murata under different initial nitrate nitrogen concentrations.

[0057] Figure 4 This is a statistical chart of the denitrification effect of Methylophaga murata under different initial nitrate nitrogen concentrations.

[0058] Figure 5 This is a statistical chart of the conversion of nitrate nitrogen by Methylophaga murata under different initial nitrate nitrogen concentrations.

[0059] Figure 6 This is a statistical table of nitrogen transformation-related functional metabolic genes carried by Methylophaga murata.

[0060] Figure 7 This is a statistical table of functional metabolic genes related to salt tolerance strategy carried by Methylophaga murata.

[0061] Figure 8 This is a statistical chart of the denitrification effect of Pseudomonas chengduensis under the same salinity. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] Methylophaga murata was deposited in Guangdong Microbiological Culture Collection Center on February 28, 2012, with the deposit number GDMCC NO: 1.508.

[0065] Pseudomonas chengduensis was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 20, 2020, with the deposit number GDMCC NO: 1.2017.

[0066] The above two strains were purchased directly from Guangdong Microbiological Culture Collection Center, and their preservation information can be directly obtained through Guangdong Microbiological Culture Collection Center.

[0067] Example 1 Preparation of Methylophaga murata pure bacterial solution

[0068] The preparation method of Methylophaga murata pure bacterial liquid comprises the following steps:

[0069] S1. Inoculate Methylophaga murata on solid culture medium and culture until a single colony grows;

[0070] S2. Pick up a single colony obtained in step S1 and perform seed culture at 30°C for 48 hours to obtain a seed solution;

[0071] S3. The seed solution obtained in step S2 is inoculated into a liquid culture medium and cultured at 30°C to obtain a Methylophaga murata bacterial solution. The absorbance of the bacterial solution at 600 nm is detected by a spectrophotometer to monitor the growth of the microorganism. 600 After the value reaches 0.8 or above, add sponge fillers and gravel particles sterilized by high pressure steam at 121°C for 20 minutes and cooled naturally into the bacterial solution. The mass ratio of sponge fillers to gravel particles is 1:2. Continue to cultivate the bacterial solution at a temperature of 30°C and a speed of 160rpm for 12 hours, and use physical adsorption to fix the microorganisms and the carrier. Among them, the mixing ratio of Methylophaga murata bacterial solution and microbial carrier is 1:18L / g.

[0072] The appropriate medium (1L, here liquid medium) for culturing Methylophaga murata contains potassium nitrate (1g), potassium dihydrogen phosphate (1g), magnesium sulfate (0.22g), sodium chloride (30g), methanol (10mL), trace element solution (1mL), vitamin B12 (20μg), and additionally prepare 2mol / L sodium bicarbonate solution and 1mol / L sodium carbonate solution, and add 50mL and 10mL to each liter of medium respectively. The trace element solution (1L) contains manganese chloride tetrahydrate (4.0g), ferrous sulfate heptahydrate (2.0g), boric acid (1.0g), calcium chloride (1.0g), cobalt chloride hexahydrate (1.6g), copper sulfate pentahydrate (1.5g), and zinc sulfate heptahydrate (3.0g). The medium was sterilized at 121°C for 20min and used after cooling to room temperature. The vitamin B12 solution is sterilized and cooled in the culture medium and then injected into the culture medium after passing through a 0.22 μm filter membrane.

[0073] The solid culture medium is a liquid culture medium with 2% (mass percentage of the liquid culture medium) agar powder added thereto.

[0074] Example 2 Experiment on the denitrification effect of Methylophaga murata on a small scale in the laboratory

[0075] 1. Experimental methods

[0076] A green and efficient microbial treatment method for removing nitrate nitrogen from high-salinity wastewater, specifically comprising the following steps:

[0077] According to step S3 of Example 1, 10 mL of bacterial liquid was obtained and inoculated into 100 mL of a suitable liquid culture medium, and nitrate nitrogen was added to the liquid culture medium in the form of potassium nitrate to make the initial nitrate nitrogen content 200 mg / L. Without adding sponge fillers and sand and gravel particles, aerobic conditions were maintained, and the culture was shaken at 30°C and a speed of 150 rpm. The effluent water sample was collected every two hours, and the extracellular nitrate nitrogen was measured after filtering through a 0.22 μm filter membrane. Among them, the nitrate nitrogen concentration was measured using phenol disulfonic acid spectrophotometry.

[0078] 2. Experimental results

[0079] The conversion of nitrate nitrogen by Methylophaga murata at an initial nitrate nitrogen concentration of 200 mg / L is shown in the figure. Figure 1 The results show that under laboratory small-scale culture conditions, microorganisms start denitrification faster and more efficiently, achieving complete removal of nitrate nitrogen in 10 hours. Even without adding fillers to enrich and fix the microorganisms, a good nitrate nitrogen removal effect can be achieved.

[0080] Example 3 Experimental study on the denitrification effect of Methylophaga murata in a reactor (large scale)

[0081] 1. Experimental methods

[0082] A green and efficient microbial treatment method for removing nitrate nitrogen from high-salinity wastewater, specifically comprising the following steps:

[0083] S1. Use the reactor already in the laboratory. The schematic diagram of the device is as follows Figure 2 As shown, a water inlet and a water outlet are set at the top, and the Methylophaga murata bacterial liquid is put into the reactor in the form of a microbial carrier prepared in Example 1, and the addition amount is 37% of the reactor volume. The total volume of the reactor is 5L, of which the liquid culture medium (the same as Example 1) is 60% of the reactor volume. A gas phase space is left at the top. The agitator circulates the water flow clockwise at 60r / min to allow the microorganisms to fully contact the culture matrix. An electric heating jacket is used to maintain the reactor temperature at 30°C during the entire operation. The reactor operates in a cycle, including a sample injection period, a reaction period, a precipitation period and a drainage period. During the water inlet period, a peristaltic pump is used to enter from the corresponding water inlet to add high-salinity wastewater to be treated. During the drainage period, the supernatant is discharged, and the pH value is regulated by adding sodium bicarbonate and potassium dihydrogen phosphate as buffers. At the same time, sodium bicarbonate and potassium dihydrogen phosphate can also be used as inorganic carbon sources and phosphorus source matrices by microorganisms.

[0084] S2. Set up three independent reactors, adjust the nitrate nitrogen concentration in the high-salinity wastewater to 10 mg / L, 100 mg / L and 200 mg / L respectively by potassium nitrate, and adjust the initial pH of the wastewater to 8 (adjusted by hydrochloric acid and sodium hydroxide). Then put the microbial carrier and bacterial liquid mixture prepared in Example 1 into the reactor, and the addition amount is 37% of the total liquid volume in the reactor. Start the reactor under aerobic conditions. During the operation of the reactor, collect effluent samples every two hours, and determine the extracellular nitrate nitrogen after filtering through a 0.22 μm filter membrane. And take 10 mL of effluent samples at 0 hours, 8 hours and 16 hours of cultivation for the determination of extracellular inorganic nitrogen and intracellular organic nitrogen. Among them, the nitrate nitrogen concentration was determined by phenoldisulfonic acid spectrophotometry; the nitrite nitrogen concentration was determined by diazo coupling spectrophotometry; the ammonium nitrogen concentration was determined by sodium reagent spectrophotometry; the intracellular organic nitrogen was determined by centrifuging the water sample, washing the precipitate three times with sterile ultrapure water and then determining it by alkaline potassium persulfate ultraviolet spectrophotometry.

[0085] 2. Experimental results

[0086] Start the reactor and adjust the initial nitrate nitrogen concentration in the initial influent to obtain the growth conditions of Methylophaga murata under different conditions. Figure 3 As shown in the figure, as the initial nitrate nitrogen concentration increases, the growth rate of microorganisms is faster, and the accumulated biomass in the final reactor is higher. When the nitrate nitrogen content does not exceed 200 mg / L, the culture time must last at least 18 hours; when the nitrate nitrogen content is greater than 200 mg / L, the culture time should be appropriately extended according to the actual detection situation.

[0087] The denitrification effect of Methylophaga murata under different initial nitrate nitrogen concentrations Figure 4 As shown, when the nitrate nitrogen concentration in the water body does not exceed 200 mg / L, the nitrate nitrogen contained in the culture medium will be completely converted into biomass nitrogen and stored in the microorganisms within 14 hours, achieving the purpose of denitrification of the water environment.

[0088] The transformation of nitrate nitrogen by Methylophaga murata under different initial nitrate nitrogen concentrations Figure 5 As shown in the figure, the assimilation and reduction of nitrate nitrogen by microorganisms goes through three stages: nitrate nitrogen → nitrite nitrogen → ammonium nitrogen. Since nitrite nitrogen may be toxic when accumulated in organisms, a small amount of nitrite nitrogen can be detected outside the cells when the initial nitrate nitrogen concentration is high. However, when the final effluent nitrate nitrogen reaches the standard, all nitrate nitrogen is converted into biomass nitrogen and stored in the microorganisms, without producing intermediate products such as nitrous oxide and nitrogen gas. At the same time, the nitrogen removed from the wastewater can be recovered by collecting biomass sediments.

[0089] Example 4 Determination of functional metabolic genes related to nitrogen transformation and salt tolerance strategies carried by Methylophaga murata

[0090] 1. Experimental methods

[0091] The Methylophaga murata genome information was downloaded from the GenBank DNA sequence database of the National Center for Biotechnology Information of the United States (GenBank number: GCF_001720165.1). The ORFs of the Methylophaga murata genome were predicted using Prodigal, and these predicted ORFs were compared with the kofam database using DIAMOND blastx (v2.0.15) for functional gene annotation.

[0092] 2. Experimental results

[0093] The inventor team found that the Methylophaga murata strain only contains complete nitrate assimilation and reduction functional genes (nasA, nasD, nasE) in multiple nitrogen conversion pathways, and has multiple nitrate transport genes (nrtA, nrtB, nrtC, nrtP), which can quickly transport nitrate nitrogen in wastewater into cells for nitrate assimilation and reduction, and will not have other nitrate nitrogen conversion pathways that may produce intermediates. It also has the functions of assimilating and reducing nitrite nitrogen (nasD, nasE) and assimilating ammonium nitrogen (amt, glnA, gltB, gltD, gdhA) in wastewater ( Figure 6 On the other hand, this strain contains genes for multiple salt tolerance strategies, including converting intracellular Na + Transport to extracellular strategy (nqrB, nqrE, nqrF), absorb extracellular K+ to maintain osmotic pressure strategy (kdpC, kup, phaA, phaC, phaG), synthesize soluble small molecule compounds to maintain osmotic pressure strategy (ectA, ectB, ectC, otsA, gdh, proA, proC, proB) Figure 7 The various salinity adaptation strategies enable the Methylophaga murata strain in the present invention to be applicable to denitrification treatment in wastewater with a wider salinity range (0.3-20 wt % in terms of sodium chloride concentration).

[0094] Comparative Example 1 A microbial treatment method for removing nitrate nitrogen from high-salinity wastewater

[0095] The difference from Example 3 is that the Methylophaga murata bacteria is replaced by the microorganism Pseudomonas chengduensis having the function of nitrate nitrogen assimilation and reduction to carry out the enrichment process as in Example 1 and the experimental process as in Example 2, ensuring that the initial influent nitrate nitrogen concentration is 200 mg / L, and the other culture conditions are the same as those in the steps of Example 2.

[0096] The results are as follows Figure 8 As shown in the figure, the denitrification rate and denitrification effect of Pseudomonas chengduensis after 16 hours were lower than those of Methylophaga murata, and a plateau was reached at 14 hours. The concentration of nitrate nitrogen in the wastewater decreased slightly as the denitrification time continued to increase.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The application of methylophage bacteria in treating high nitrate nitrogen wastewater is characterized in that: The methylophaga is Methylophaga murata, which was deposited in Guangdong Microbiological Culture Collection Center on February 28, 2012, with the deposit number of GDMCC NO: 1.

508.

2. A microbial treatment method for removing nitrate nitrogen from high-salinity wastewater, characterized in that: The following steps are involved: The pH value of the wastewater containing nitrate nitrogen is adjusted to 6-9, and the Methylophaga murata bacterial solution is inoculated therein, and the mixture is fully mixed to carry out microbial denitrification under aerobic conditions; Wherein, the Methylophaga murata bacterial liquid is attached to a microbial carrier or directly added into the nitrate-containing wastewater; The methylophaga is Methylophaga murata, which was deposited in Guangdong Microbiological Culture Collection Center on February 28, 2012, with the deposit number of GDMCC NO: 1.

508.

3. The microbial treatment method according to claim 2, characterized in that: The OD of the Methylophaga murata bacterial solution 600 Value ≥ 0.

8.

4. The microbial treatment method according to claim 2, characterized in that: The OD of the Methylophaga murata bacterial solution after being added into the wastewater containing nitrate nitrogen is 600 Value ≤ 0.

1.

5. The microbial treatment method according to claim 2, characterized in that: Calculated by volume percentage of the wastewater containing nitrate nitrogen, the inoculation amount of the Methylophaga murata bacterial solution is 5% to 40%.

6. The microbial treatment method according to any one of claims 2 to 5, characterized in that: The preparation method of the Methylophagamurata bacterial liquid comprises the following steps: S1. Inoculate Methylophaga murata on solid culture medium and culture until a single colony grows; S2. Pick up the single colony obtained in step S1 for seed culture to obtain seed solution; S3. The seed solution obtained in step S2 is inoculated into a liquid culture medium for expansion culture to obtain a Methylophagamurata bacterial solution.

7. The microbial treatment method according to any one of claims 2 to 5, characterized in that: The preparation method of the Methylophagamurata bacterial liquid comprises the following steps: S1. Inoculate Methylophaga murata on solid culture medium and culture until a single colony grows; S2. Pick up the single colony obtained in step S1 for seed culture to obtain seed solution; S3. Inoculate the seed solution obtained in step S2 into the liquid culture medium for expansion culture; when the OD of the bacterial solution 600 When the value is ≥0.8, a sterilized microbial carrier is added to the bacterial solution, and the culture is continued so that the Methylophaga murata bacterial solution is fully attached to the microbial carrier to obtain the Methylophaga murata bacterial solution.

8. The microbial treatment method according to claim 2, characterized in that: The microbial carrier includes one or more of sponge filler, sand and gravel particles, and activated carbon.

9. The microbial treatment method according to claim 2, characterized in that: The nitrate-nitrogen-containing wastewater includes nutrients.

10. The microbial treatment method according to claim 2, characterized in that: The temperature of the microbial denitrification is 20-32°C.

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