Method for separating and purifying sulfur autotrophic denitrifying bacteria in coking sewage and application

Abstract

The invention provides a method for separating and purifying sulfur autotrophic denitrifying bacteria in coking sewage and application, and belongs to the technical field of sewage treatment.The method comprises the steps that sludge in an anoxic pond of a coking sewage plant is taken and inoculated into an enrichment culture medium, enrichment culture is conducted, an obtained enrichment culture is diluted and then coated to an isolation culture medium, and the sulfur autotrophic denitrifying bacteria are obtained through isolation culture. The sulfur autotrophic denitrifying bacteria are obtained; the sulfur autotrophic denitrifying bacteria are used for treating wastewater. The method disclosed by the invention has the advantages that the process is simple, and only 4-5 days are needed for operating the preparation of the squaring bacteria and the isolation culture medium to the purification culture; the method is high in separation and purification success rate, and the purified culture can be obtained through one-time separation and purification. Therefore, the sulfur autotrophic denitrifying bacteria can be widely applied to a sewage denitrification process.

Description

Technical Field

[0001] The invention relates to the technical field of sewage treatment, and in particular to a method for separating and purifying sulfur autotrophic denitrifying bacteria in coking sewage and application thereof. Background Art

[0002] The coking process is a key step in converting coal into coke and other chemical products by high-temperature heating. A large amount of coking wastewater is generated in this process. Coking wastewater contains high concentrations of organic pollutants (such as benzene, phenolic compounds and polycyclic aromatic hydrocarbons), inorganic salts (such as sulfides, cyanides and ammonia nitrogen) and heavy metal ions. These pollutants are not only highly toxic, but also have poor biodegradability, pose significant environmental risks, and are extremely difficult to treat. Traditional sewage treatment methods often have limited effects on coking wastewater, especially for nitrogen oxides and sulfur-containing compounds, which still face great challenges.

[0003] In the sewage treatment process, nitrification and denitrification are two key steps for nitrogen removal. Nitrification converts ammonia nitrogen into nitrate, while denitrification reduces nitrate to nitrogen gas to achieve the purpose of nitrogen removal. However, the traditional denitrification process requires the addition of an organic carbon source (such as methanol, acetic acid, etc.) as an electron donor to maintain the metabolic activity of denitrifying bacteria. This external carbon source not only increases the treatment cost, but may also cause excessive residual organic matter in the effluent due to improper control of the carbon source dosage, thereby causing secondary pollution. In addition, when treating coking wastewater, due to its high concentration of toxic and harmful substances, the activity of traditional denitrifying bacteria is often inhibited, resulting in poor treatment effect.

[0004] Unlike traditional heterotrophic denitrifying bacteria, sulfur autotrophic denitrifying bacteria can use inorganic sulfides (such as hydrogen sulfide, sulfur, and thiosulfate) as electron donors to reduce nitrate and nitrite to nitrogen gas. This sulfur autotrophic denitrification process does not require an external organic carbon source and has the advantages of being economical, efficient, and environmentally friendly. Sulfur autotrophic denitrifying strains have strong tolerance and metabolic capacity for sulfides in coking wastewater, and are therefore considered to be ideal microorganisms for treating sulfur-containing industrial wastewater. However, despite the huge potential of this technology, it still faces multiple challenges in practical applications, especially in the separation and purification of efficient strains. Summary of the invention

[0005] In view of the above problems, the present invention provides a method and application for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater, and screens out sulfur autotrophic denitrifying strains with high tolerance and high nitrate reduction ability by optimizing the separation medium formula and regulating environmental conditions. The present invention not only helps to improve the treatment effect of coking wastewater, but also will provide important theoretical support and technical reserves for the industrial application of sulfur autotrophic denitrification technology.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater, characterized in that the method comprises the following specific steps:

[0008] S1. Enrichment culture of sulfur autotrophic denitrifying bacteria

[0009] The sludge in the anoxic tank of the coking wastewater plant is inoculated into the enrichment medium, and the enrichment culture is obtained by enrichment culture;

[0010] S2. Isolation and culture of sulfur autotrophic denitrifying bacteria

[0011] The enriched culture is diluted and then applied to a separation culture medium. After separation and culture, a separation culture is obtained, which is the sulfur autotrophic denitrifying bacteria.

[0012] Further, in step S1, the formula of the enrichment medium is 0.90-1.10 mg / L sodium bicarbonate, 0.65-0.75 mg / mL sodium succinate, 0.90-1.10 mg / mL ammonium acetate, 0.46-0.48 mg / mL sodium thiosulfate, 0.18-0.22 mg / mL potassium nitrate and 48-52 mL / L Weiss salt solution;

[0013] In step S2, the formula of the separation medium is potassium nitrate 0.90-1.10 mg / mL, sodium thiosulfate 0.90-1.10 mg / mL, sodium bicarbonate 0.45-0.55 mg / mL, ammonium acetate 0.65-0.75 mg / mL, 0.1% bromothymol blue solution 2.8-3.2 mL / L, trisodium citrate 7.2-7.4 mg / mL, potassium dihydrogen phosphate 0.90-1.10 mg / mL, magnesium sulfate heptahydrate 0.90-1.10 mg / mL, anhydrous calcium chloride 0.18-0.22 mg / mL, ferrous sulfate heptahydrate 0.05-0.07 mg / mL, trace element solution 9-11 mL / L and agar 2.5-2.7 mg / mL, and the pH is adjusted to 6.5-7.5 with acid and alkali.

[0014] Further, in the formula of the enrichment medium, the formula of Wei's salt solution is 4.80-5.20 mg / mL of potassium dihydrogen phosphate, 2.4-2.6 mg / mL of magnesium sulfate heptahydrate, 2.4-2.6 mg / mL of sodium chloride, 0.04-0.06 mg / mL of ferrous sulfate heptahydrate, 0.04-0.06 mg / mL of manganese sulfate heptahydrate and 1000 mL of distilled water;

[0015] In the formula of the separation medium, the formula of the trace element solution is 4.8-5.2 mg / mL of ethylenediaminetetraacetic acid, 0.0018-0.0022 mg / mL of boric acid, 0.0035-0.0045 mg / mL of zinc sulfate, 0.008-0.012 mg / mL of manganese sulfate and 0.045-0.055 mg / mL of anhydrous copper sulfate.

[0016] Further, in step S1, the enrichment medium is prepared by adding the enrichment medium formula to distilled water for dissolution, adjusting the pH to 6.5-7.5, dispensing into glass tissue culture bottles, adding 3 glass beads to enhance stirring and break up the mud sample, adding a tissue culture cover and tightly placing in boiling water for 30-40 minutes for sterilization, thereby obtaining the enrichment medium;

[0017] In step S2, the separation medium is prepared by dissolving it in distilled water according to the formula of the separation medium, adjusting the pH to 6.5-7.5, cooling it until there is no obvious heat emission, and then dispensing it into a glass flat culture dish, dispensing 10 mL, half-covering the culture dish with the culture medium and the culture dish with a large lid, placing it in a high-temperature sterilization oven, heating it at 103°C for 60 minutes, and sterilizing it to obtain the separation medium.

[0018] Further, in step S1, the mass ratio of sludge to enrichment medium is 1:40-50;

[0019] Enrichment culture is a constant temperature shaking culture at 25-35°C;

[0020] In step S2, the separation culture is carried out at a constant temperature of 28 to 40°C.

[0021] Furthermore, the method also includes the purification and cultivation of sulfur autotrophic denitrifying bacteria, as follows:

[0022] Use a 1 mL sterile syringe to draw 500 μL of the colonies with obvious blue changes around them in the isolated culture, inject it into 75 mL of the purification culture medium, and culture at a constant temperature of 30 to 45°C until the liquid of the purification culture medium becomes turbid visually, thereby obtaining a purified culture;

[0023] The purified culture was inoculated into the purification medium again for further separation and purification to obtain the culture. The OD of the purified product was 600 Greater than 1;

[0024] The purified culture and the culture are both sulfur autotrophic denitrifying bacteria.

[0025] Further, the formula of the purification medium is potassium nitrate 0.90-1.10 mg / mL, sodium thiosulfate 0.90-1.10 mg / mL, sodium bicarbonate 0.45-0.55 mg / mL, ammonium acetate 0.65-0.75 mg / mL, trisodium citrate 7.2-7.4 mg / mL, potassium dihydrogen phosphate 0.90-1.10 mg / mL, magnesium sulfate heptahydrate 0.90-1.10 mg / mL, anhydrous calcium chloride 0.18-0.22 mg / mL, ferrous sulfate heptahydrate 0.05-0.07 mg / mL and trace element solution 9-11 mL / L, and the pH is adjusted to 6.5-7.5 with acid and alkali;

[0026] The formula of the trace element solution is 4.8-5.2 mg / mL of ethylenediaminetetraacetic acid, 0.0018-0.0022 mg / mL of boric acid, 0.0035-0.0045 mg / mL of zinc sulfate, 0.008-0.012 mg / mL of manganese sulfate, and 0.045-0.055 mg / mL of anhydrous copper sulfate;

[0027] The preparation method of the purified culture medium is to dissolve it in distilled water according to the formula of the purified culture medium, adjust the pH to 6.5-7.5, and then divide it into glass flat culture dishes after cooling until there is no obvious heat emission, divide it into 10 mL, half-cover the culture dish with the culture medium and the culture dish with a large lid, place it in a high-temperature sterilization oven, heat it at 103°C for 60 minutes, sterilize it, and obtain the purified culture medium.

[0028] Application of sulfur autotrophic denitrifying bacteria obtained by the method in treating wastewater.

[0029] Furthermore, sulfur autotrophic denitrifying bacteria are added to the wastewater and shaken for denitrification treatment.

[0030] Furthermore, during the denitrification process, the volume ratio of sulfur autotrophic denitrifying bacteria to wastewater was 0.8-1.2:100;

[0031] The shaking temperature is 30-45°C and the time is more than 24 hours.

[0032] The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater of the present invention and the beneficial effects of the application thereof are:

[0033] The sulfur autotrophic denitrifying bacteria separated and purified by the present invention are bacteria species that use nitrate as an electron acceptor and one or more reduced or partially reduced sulfur-containing compounds as an energy source under anaerobic conditions;

[0034] The sulfur autotrophic denitrifying bacteria of the present invention are strictly autotrophic in the mode of using inorganic electron donors and only carbon dioxide as their main carbon source, but many or all species can assimilate certain organic compounds if thiosulfate or other inorganic electron donors are provided as energy sources;

[0035] The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater of the present invention has simple process, convenient operation, easy-to-obtain materials, strong pertinence, and low requirements on operators and equipment;

[0036] The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater of the present invention has a fast separation and purification speed, and only takes 4 to 5 days from the preparation of sulfur autotrophic denitrifying bacteria and separation culture medium to the purification of culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a morphological diagram of the enrichment culture cultured for the first time on the separation medium in Example 1 of the present invention; wherein, no foreign bacteria grow around the enrichment culture cultured in the enrichment medium when inoculated into the separation medium;

[0038] Figure 2 This is a morphological diagram of the enrichment culture cultured for the second time on the separation medium in Example 1 of the present invention; wherein, there is a blue change around the colonies on the separation medium;

[0039] Figure 3 is a morphological diagram of the separation culture obtained by the third plate streak culture on the separation medium in Example 1 of the present invention;

[0040] Figure 4 is the evolutionary tree of the isolated culture in Example 1 of the present invention;

[0041] Figure 5 It is the NO after the culture treats wastewater in Example 1 of the present invention. 3- with NO 2- Mass concentration variation diagram of

[0042] Figure 6 The sulfate, ammonia nitrogen, and NO in the wastewater treated by the culture in Example 1 of the present invention are 3- 、NO 2- Percentage content change diagram;

[0043] Figure 7 is the OD of the culture after treating wastewater in Example 1 of the present invention 600 And CODcr change graph. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0045] Example 1 A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater and its application

[0046] This embodiment relates to a method and application of separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater, wherein in the processes of enrichment culture, separation culture, purification culture and further purification, all vessels involved, including but not limited to glass tubes, culture dishes, etc., are sterilized at 103-105° C. for 80 minutes before use. The specific method and application process are as follows:

[0047] S1. Enrichment culture of sulfur autotrophic denitrifying bacteria

[0048] S11. Preparation of enrichment medium

[0049] The formula of the enrichment medium is sodium bicarbonate 1.00 mg / mL, sodium succinate 0.7 mg / mL, ammonium acetate 1.00 mg / mL, sodium thiosulfate 0.47 mg / mL, potassium nitrate 0.20 mg / mL, and Wei's salt solution 50 mL / L.

[0050] Dissolve 5.00 mg / mL of potassium dihydrogen phosphate, 2.5 mg / mL of magnesium sulfate heptahydrate, 2.5 mg / mL of sodium chloride, 0.05 mg / mL of ferrous sulfate heptahydrate, 0.05 mg / mL of manganese sulfate heptahydrate and 1000 mL of distilled water to prepare a Vickers salt solution.

[0051] Then add it to distilled water according to the enrichment medium formula to dissolve, adjust the pH to 6.5-7.5 (the adjusted pH value is 6.8 in this embodiment), and dispense it into 100 mL glass tissue culture bottles, dispense 45 mL, make a group of three parallel and one blank, put 3 glass beads to enhance stirring and break up the mud sample, a total of three groups, add tissue culture cover and place it tightly in boiling water for 30-40 minutes for sterilization (sterilized for 30 minutes in this embodiment) to obtain enrichment medium.

[0052] S12, enrichment culture

[0053] A 100 mL volumetric flask is used to measure 100 mL of the mixed solution in the anoxic tank of the coking sewage plant. After settling for 1 hour, the supernatant is skimmed off, and the sludge at the bottom of the volumetric flask is taken, and injected into the above-prepared enrichment medium at a mass ratio of 1:40, and cultured at a constant temperature of 25 to 35 ° C. After the first shaking culture for 8 hours, 5 mL of the bacterial solution is inoculated into a fresh enrichment medium, and the second constant temperature shaking culture is carried out at 25 to 35 ° C. for 10 hours, and then 5 mL of the bacterial solution is inoculated into a fresh enrichment medium, and the third constant temperature shaking culture is carried out at 25 to 35 ° C. for 22 hours. After the third constant temperature shaking culture is completed, an enrichment culture is obtained. The temperature of the three constant temperature shaking cultures in this embodiment is 32 ° C.

[0054] Among them, the mixed liquid collected from the anoxic tank of the coking wastewater plant was sealed and stored at 4°C for no more than 3 days.

[0055] S2. Isolation and culture of sulfur autotrophic denitrifying bacteria

[0056] S21. Preparation of separation medium

[0057] The formula of the separation medium is potassium nitrate 1.0 mg / mL, sodium thiosulfate 1.0 mg / mL, sodium bicarbonate 0.5 mg / mL, ammonium acetate 0.7 mg / mL, 0.1% bromothymol blue solution 3 mL / L, trisodium citrate 7.3 mg / mL, potassium dihydrogen phosphate 1.00 mg / mL, magnesium sulfate heptahydrate 1.00 mg / mL, anhydrous calcium chloride 0.2 mg / mL, ferrous sulfate heptahydrate 0.06 mg / mL, trace element solution 10 mL / L, agar 2.6 mg / mL, and the pH is adjusted to 6.5-7.5 with acid and alkali (the pH value is 7.3 in this embodiment).

[0058] According to the ratio of EDTA (ethylenediaminetetraacetic acid) 5 mg / mL, boric acid 0.002 mg / mL, zinc sulfate 0.004 mg / mL, manganese sulfate 0.01 mg / mL and anhydrous copper sulfate 0.05 mg / mL, the above inorganic salts were dissolved in distilled water to prepare a trace element solution.

[0059] The 0.1% bromothymol blue solution is prepared by diluting bromothymol blue with anhydrous ethanol to a mass percentage concentration of bromothymol blue of 0.1%.

[0060] Then, according to the formula of the separation medium, add it to distilled water to dissolve, adjust the pH to 6.5-7.5 (the pH value in this embodiment is 7.3), cool it until there is no obvious heat emission, and then dispense it into a glass flat culture dish, dispense 10 mL, half-cover the culture dish and the culture dish with the culture medium, place it in a high-temperature sterilization oven, heat it at 103°C for 60 minutes, sterilize, and obtain the separation medium.

[0061] S22. Isolation and culture

[0062] Take 0.5 mL of the enrichment culture and dilute it to 10 in a sterilized glass tube using distilled water according to the 10-fold serial dilution method. -3 ~10 -4 10 -3 ~10 -4 Gradient dilutions (10 -3 and 10 -4 The two gradient dilutions were subsequently applied), and then the dilutions were applied to the separation culture medium, and then placed in a constant temperature incubator for culture at a temperature of 28-40°C. After 24 hours of culture, no bacteria were found growing around the enrichment culture. Figure 1 Observe the morphology of the colonies on the separation medium. There is no obvious difference in the colonies. The colonies have neat edges, no bacteria grow around them, are opaque, and have a blue color change around the separation medium. Replace the separation medium and continue to culture in a constant temperature incubator at 28-40℃ until the separation medium has a blue color change. Figure 2 As shown. Pick the strains with blue changes around them and continue to streak on the separation medium. Replace the separation medium again and continue to culture in a constant temperature incubator at 28-40°C for 24 hours. Separate three times in total to obtain the isolated culture, which is the sulfur autotrophic denitrifying bacteria. Figure 3 The culture temperature for the three separation cultures in this example was 32°C.

[0063] The isolated culture was sent to the cooperative unit for gene sequencing. The 16srDNA gene sequence was as follows: Figure 4 (YL270-01-16s-F evolutionary tree), the length is 1.2kbp, as follows:

[0064] :YL270-01-16s-F

[0065] The obtained 16s rDNA gene sequence of the strain was searched by BLAST in the gene database. The comparison result showed that the strain had the closest homology to Pseudomonas abyssi strain MT5 and Pseudomonas ceruminis strain BML-PP028, among which the homology to Pseudomonas ceruminis strain BML-PP028 was 100%, which confirmed that the culture belonged to a non-model engineered strain of Pseudomonas, that is, Pseudomonas genus.

[0066] S3. Purification and culture of sulfur autotrophic denitrifying bacteria

[0067] S31. Preparation of purification medium

[0068] The formula of the purification medium is potassium nitrate 1.0 mg / mL, sodium thiosulfate 1.0 mg / mL, sodium bicarbonate 0.5 mg / mL, ammonium acetate 0.7 mg / mL, trisodium citrate 7.3 mg / mL, potassium dihydrogen phosphate 1.00 mg / mL, magnesium sulfate heptahydrate 1.00 mg / mL, anhydrous calcium chloride 0.2 mg / mL, ferrous sulfate heptahydrate 0.06 mg / mL and trace element solution 10 mL / L, and the pH is adjusted to 6.5-7.5 (the pH value in this embodiment is 7.3) with acid and alkali. That is, the purification medium is obtained by optimizing the separation medium, and the only difference is that the purification medium does not add agar and 0.1% bromothymol blue solution. Since the separation medium has provided sufficient nutrients required for the growth of the separated bacteria, such as sulfur source, carbon source, nitrate, etc., and bromothymol blue will inhibit bacterial growth over a long period of time, there is no need to add 0.1% bromothymol blue solution; and since the purification medium is liquid, there is no need to add agar.

[0069] According to the ratio of EDTA (ethylenediaminetetraacetic acid) 5 mg / mL, boric acid 0.002 mg / mL, zinc sulfate 0.004 mg / mL, manganese sulfate 0.01 mg / mL and anhydrous copper sulfate 0.05 mg / mL, the above inorganic salts were dissolved in distilled water to prepare a trace element solution.

[0070] Then, according to the formula of the purified culture medium, add it to distilled water to dissolve, adjust the pH to 6.5-7.5 (the pH value in this embodiment is 7.3), cool it until there is no obvious heat emission, and then dispense it into a glass flat culture dish, dispense 10 mL, half-cover the culture dish with the culture medium and the culture dish with a large lid, place it in a high-temperature sterilization oven, heat it at 103°C for 60 minutes, sterilize it, and obtain the purified culture medium.

[0071] S32, purification and culture

[0072] 500 μL of the colonies with obvious blue changes around them in the isolated culture were sucked in with a 1 mL sterile syringe, injected into 75 mL of the purified culture medium, and placed in a constant temperature incubator for cultivation at a temperature of 30 to 45° C. (the culture temperature in this embodiment was 38° C.) until the liquid of the purified culture medium was visually turbid. At this time, OD 600 was 1.8, and purified culture was obtained.

[0073] S4. Further separation and purification of sulfur autotrophic denitrifying bacteria

[0074] The purified culture was inoculated into the purification medium again for further separation and purification to obtain the culture, OD 600 is 1.27.

[0075] S5. Application (i.e. wastewater treatment)

[0076] The effluent from the secondary sedimentation tank after primary treatment by the AO process of a coking wastewater plant (hereinafter referred to as sewage) was taken, and the primary indicators of ammonia nitrogen, nitrate nitrogen, nitrite, and CODcr were measured. The results are as follows:

[0077] Table 1 List of primary indicators in the secondary sedimentation water

[0078] Ammonia nitrogen (mg / L) Nitrate nitrogen (mg / L) Nitrite (mg / L) CODcr(mg / L) 7 50 0 289

[0079] The wastewater after the test is dosed with drugs, and a certain amount of sodium thiosulfate, ammonium chloride, potassium nitrate and sodium succinate are added to make the prepared wastewater indicators as follows:

[0080] Table 2 List of configured wastewater indicators

[0081] Ammonia nitrogen (mg / L) Nitrate nitrogen (mg / L) Nitrite (mg / L) CODcr(mg / L) 55 300 0 330

[0082] The prepared wastewater was placed in a glass beaker sterilized at 120℃ for 30min. The culture was added to the prepared wastewater at a volume ratio of 1:100. After shaking and culturing at 30℃ for 24h, samples were taken every 12h to measure OD. 600 , ammonia nitrogen, NO 3- 、NO 2- , sulfate and CODcr parameters were used to analyze the denitrification performance of the culture. The results are shown in Figure 5 to Figure 7 .

[0083] Depend on Figure 5 Medium NO 3- with NO 2- It can be seen from the change in mass concentration that a small amount of nitrite is produced after 24 hours of shaking culture, which may be the accumulation of initial nitrate nitrogen converted into nitrite nitrogen. After 48 hours, the mass concentration of nitrate nitrogen decreases significantly. After 84 hours, the mass concentrations of nitrate nitrogen and nitrite nitrogen drop to below 10 mg. After 84 hours, the removal rate of (sub)nitrite nitrogen reaches more than 96.6%, which shows that the culture obtained by the present invention has a good nitrate nitrogen removal ability.

[0084] Depend on Figure 6 Sulfate, ammonia nitrogen, NO 3- 、NO 2- The percentage and inoculation time showed that after 24 hours, ammonia nitrogen and NO 3- with NO 2- The total percentage is 90%, and after 84 hours, ammonia nitrogen and NO 3- with NO 2-The total percentage of is 15%. According to the law of conservation of matter, it can be known that the reduced part is produced and overflowed by nitrogen. And the gradual increase in the proportion of sulfate radicals indicates that a sulfur oxidation reaction has occurred. It can be shown that the culture obtained by the present invention not only has the ability to remove nitrate nitrogen, but also has the ability to oxidize sulfur and other sulfides into sulfate radicals.

[0085] Depend on Figure 7 Medium OD 600 From the relationship with CODcr, it can be seen that there is no obvious consumption of COD during the growth of microorganisms after inoculation and culture, which shows that the microorganisms in the culture of the present invention are strictly autotrophic and do not need to provide additional carbon source.

[0086] Combination Figure 5 to Figure 7 It can be seen that during the denitrification process under anaerobic conditions, the culture of the present invention is strictly autotrophic if it is supplied with sulfur or other thio compounds, and can obtain the ability to grow continuously in the coking system, and can effectively remove nitrate nitrogen, sulfur and other sulfide substances in the coking wastewater, providing a new treatment method for coking wastewater treatment.

[0087] Embodiment 2-5 A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater and its application

[0088] Examples 2 to 5 are respectively a method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater and its application. According to the optimized method and application given in Example 1, the scope of the method and application that can achieve better separation and purification of sulfur autotrophic denitrifying bacteria is further explored to obtain the methods and applications in Examples 2 to 5. The methods and applications for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater in Examples 2 to 5 are basically the same as those in Example 1, except that the dosage and some process parameters are different. See Table 3 for details:

[0089] Table 3 List of analysis parameters in Examples 2 to 5

[0090]

[0091]

[0092] The other parts of Examples 2 to 5 are the same as those of Example 1 and will not be described again.

[0093] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater, characterized in that: The method comprises the following specific steps: S1. Enrichment culture of sulfur autotrophic denitrifying bacteria The sludge in the anoxic tank of the coking wastewater plant is inoculated into the enrichment medium, and the enrichment culture is obtained by enrichment culture; S2. Isolation and culture of sulfur autotrophic denitrifying bacteria The enriched culture is diluted and then applied to a separation culture medium. After separation and culture, a separation culture is obtained, which is the sulfur autotrophic denitrifying bacteria.

2. The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to claim 1, characterized in that: In step S1, the formula of the enrichment medium is 0.90-1.10 mg / L sodium bicarbonate, 0.65-0.75 mg / mL sodium succinate, 0.90-1.10 mg / mL ammonium acetate, 0.46-0.48 mg / mL sodium thiosulfate, 0.18-0.22 mg / mL potassium nitrate and 48-52 mL / L Weiss salt solution; In step S2, the formula of the separation medium is potassium nitrate 0.90-1.10 mg / mL, sodium thiosulfate 0.90-1.10 mg / mL, sodium bicarbonate 0.45-0.55 mg / mL, ammonium acetate 0.65-0.75 mg / mL, 0.1% bromothymol blue solution 2.8-3.2 mL / L, trisodium citrate 7.2-7.4 mg / mL, potassium dihydrogen phosphate 0.90-1.10 mg / mL, magnesium sulfate heptahydrate 0.90-1.10 mg / mL, anhydrous calcium chloride 0.18-0.22 mg / mL, ferrous sulfate heptahydrate 0.05-0.07 mg / mL, trace element solution 9-11 mL / L and agar 2.5-2.7 mg / mL, and the pH is adjusted to 6.5-7.5 with acid and alkali.

3. The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to claim 2, characterized in that: In the formula of the enrichment medium, the formula of Wei's salt solution is 4.80-5.20 mg / mL of potassium dihydrogen phosphate, 2.4-2.6 mg / mL of magnesium sulfate heptahydrate, 2.4-2.6 mg / mL of sodium chloride, 0.04-0.06 mg / mL of ferrous sulfate heptahydrate, 0.04-0.06 mg / mL of manganese sulfate heptahydrate and 1000 mL of distilled water; In the formula of the separation medium, the formula of the trace element solution is 4.8-5.2 mg / mL of ethylenediaminetetraacetic acid, 0.0018-0.0022 mg / mL of boric acid, 0.0035-0.0045 mg / mL of zinc sulfate, 0.008-0.012 mg / mL of manganese sulfate and 0.045-0.055 mg / mL of anhydrous copper sulfate.

4. A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to claim 2 or 3, characterized in that: In step S1, the enrichment medium is prepared by adding the enrichment medium formula into distilled water to dissolve, adjusting the pH to 6.5-7.5, breaking, and sterilizing to obtain the enrichment medium; In step S2, the separation medium is prepared by adding the separation medium according to the formula of the separation medium into distilled water for dissolution, adjusting the pH to 6.5-7.5, cooling, and sterilizing to obtain the separation medium.

5. A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to any one of claims 1 to 3, characterized in that: In step S1, the mass ratio of sludge to enrichment medium is 1:40-50; Enrichment culture is a constant temperature shaking culture at 25-35°C; In step S2, the separation culture is carried out at a constant temperature of 28 to 40°C.

6. A method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to any one of claims 1 to 3, characterized in that: The method also includes the purification and cultivation of sulfur autotrophic denitrifying bacteria, which is specifically as follows: The separated culture is injected into the purification medium, and cultured at a constant temperature of 30 to 45° C. until the liquid of the purification medium becomes turbid visually, thereby obtaining a purified culture; The purified culture was inoculated into the purification medium again for further separation and purification to obtain the culture. The OD of the purified product was 600 Greater than 1; The purified culture and the culture are both sulfur autotrophic denitrifying bacteria.

7. The method for separating and purifying sulfur autotrophic denitrifying bacteria from coking wastewater according to claim 6, characterized in that: The formula of the purification medium is potassium nitrate 0.90-1.10 mg / mL, sodium thiosulfate 0.90-1.10 mg / mL, sodium bicarbonate 0.45-0.55 mg / mL, ammonium acetate 0.65-0.75 mg / mL, trisodium citrate 7.2-7.4 mg / mL, potassium dihydrogen phosphate 0.90-1.10 mg / mL, magnesium sulfate heptahydrate 0.90-1.10 mg / mL, anhydrous calcium chloride 0.18-0.22 mg / mL, ferrous sulfate heptahydrate 0.05-0.07 mg / mL and trace element solution 9-11 mL / L, and the pH is adjusted to 6.5-7.5 with acid and alkali; The formula of the trace element solution is 4.8-5.2 mg / mL of ethylenediaminetetraacetic acid, 0.0018-0.0022 mg / mL of boric acid, 0.0035-0.0045 mg / mL of zinc sulfate, 0.008-0.012 mg / mL of manganese sulfate, and 0.045-0.055 mg / mL of anhydrous copper sulfate; The preparation method of the purified culture medium is to add the purified culture medium into distilled water to dissolve it according to the formula of the purified culture medium, adjust the pH to 6.5-7.5, and sterilize it to obtain the purified culture medium.

8. Use of sulfur autotrophic denitrifying bacteria obtained by the method according to any one of claims 1 to 7 in treating wastewater.

9. The use according to claim 8, characterized in that: Sulfur autotrophic denitrifying bacteria are added to the wastewater and shaken for denitrification.

10. The use according to claim 9, characterized in that: During the denitrification process, the volume ratio of sulfur autotrophic denitrifying bacteria to wastewater is 0.8-1.2:100; The shaking temperature is 30-45°C and the time is more than 24 hours.

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