Screening culture medium and rapid screening method for oligotrophic HN-AD bacteria

Abstract

The invention provides a screening culture medium and a rapid screening method of oligotrophic HN-AD bacteria, and belongs to the technical field of microorganisms. The rapid screening method comprises the following steps: enriching oligotrophic bacteria by adopting a low-nutrition formula and carrying out primary screening; adopting a 96-well plate and utilizing HNM2, DM1 and DM2 culture media to carry out naked eye rapid re-screening in sequence, so as to obtain the oligotrophic HN-AD strain with strong denitrification (nitrification and denitrification) capability; the N8 strain capable of efficiently removing nitrogen pollution in a water body with a low carbon-nitrogen ratio is verified by adopting a rapid screening method. The rapid screening method is high in selectivity, high in accuracy, simple and rapid, is very suitable for screening of a large batch of samples, and may become a rapid screening method with a great application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a screening culture medium and a rapid screening method for oligotrophic HN-AD bacteria. Background Art

[0002] Nitrogen pollutants in water bodies mainly include ammonia nitrogen (NH4 + -N / NH3), nitrite nitrogen (NO 2- -N) and nitrate nitrogen (NO 3- -N). High concentrations of nitrogen pollution not only cause eutrophication of water bodies, harm aquatic life, and destroy ecological balance, but also seriously threaten the health and safety of humans and aquatic animals. In recent years, the efficient removal of nitrogen-containing pollutants in water bodies has become a global research hotspot.

[0003] Microbial technology has the advantages of safety, environmental protection, economy, and high efficiency. It is currently one of the more ideal technologies for in-situ reduction of water pollutants. Traditional biological denitrification technology uses ammonia oxidizing bacteria and nitrite oxidizing bacteria as autotrophic aerobic bacteria, and denitrifying bacteria as heterotrophic anaerobic bacteria. It is necessary to separate nitrification and denitrification. Its operating cost is high, the process is complicated and the action time is long. It is greatly affected by external factors and is prone to secondary pollution. The discovery of heterotrophic nitrification-aerobic denitrifying (HN-AD) bacteria effectively solves the problem of nitrification and denitrification zoning. This type of bacteria has the characteristics of simultaneous nitrification and denitrification. Under aerobic conditions, ammonia nitrogen and total nitrogen can be removed simultaneously in one reactor, which to a certain extent makes up for the shortcomings of traditional biological denitrification that is limited by oxygen.

[0004] At present, many bacterial genera with HN-AD capabilities have been isolated from various environments, and the carbon-nitrogen ratio is one of the key parameters affecting the denitrification efficiency of HN-AD. At present, low carbon-nitrogen ratios are common in surface water (rivers, lakes, reservoirs, etc.), urban sewage and aquaculture wastewater. It is generally believed that sewage with COD / TN < 8 (or BOD / TN < 5) is low carbon-nitrogen ratio sewage. Due to the serious lack of carbon source energy in low carbon-nitrogen ratio wastewater, it is difficult to achieve the expected effect of nitrogen removal using HN-AD. Therefore, it is particularly important to screen out HN-AD bacteria suitable for the removal of nitrogen pollution in low carbon-nitrogen ratio water bodies.

[0005] Oligotrophic bacteria are a type of microorganism that grows in an environment with very low nutrient concentrations, and are usually defined as bacteria that can grow on a medium containing 1-15 mg / L carbon during initial culture. This type of bacteria has stable special functions and genetic genes, low demand for nutrients, and strong denitrification and environmental tolerance in oligotrophic environments, so it has unique advantages when treating low C / N ratio water bodies. In the prior art, most HN-AD strains are enriched and screened using high-nutrient culture media, which is a time-consuming and labor-intensive process, and the isolated strains are not suitable for treating low-carbon-nitrogen ratio water bodies. Therefore, establishing a fast, efficient, and targeted screening method for low-carbon oligotrophic HN-AD bacteria is a technical problem that needs to be solved at this stage. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a screening culture medium and a rapid screening method for oligotrophic HN-AD bacteria. The strains screened by this method can effectively remove nitrogen pollution in water bodies with a low carbon-nitrogen ratio, improve the treatment efficiency of nitrogen pollution in water bodies with a low carbon-nitrogen ratio, and reduce costs.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The invention provides a screening culture medium for oligotrophic HN-AD bacteria, comprising an HNM culture medium, a DM culture medium and a BTB culture medium. The HNM culture medium formula comprises: (NH4)2SO4 0.014-0.47g, trisodium citrate 0.053-1.80g, K2HPO4·3H2O 0.10-0.30g, MgSO4·7H2O 0.03-0.07g, NaCl 0.08-0.16g, FeSO4·7H2O 0.005-0.015g, MnSO4·H2O 0.005-0.015g, CaCl2 0.03-0.07g, 1000mL of distilled water, and the pH value is adjusted to 6.8-7.2; the DM culture medium formula comprises: KNO3 0.5-0.72g, trisodium citrate 1.60-2.0g, K2HPO4·3H2O 0.10-0.30g, MgSO4·7H2O0.03-0.07g, NaCl 0.08-0.16g, FeSO4·7H2O 0.005-0.015g, MnSO4·H2O 0.005-0.015g, CaCl2 0.03-0.07g, distilled water 1000mL, adjusted pH to 6.8-7.2; BTB culture medium formula includes: trisodium citrate 1.60-2.00g; NaNO3 0.51-0.71g; KH2PO4 0.50-1.50g; FeSO4·7H2O 0.10-0.30g; MnSO4·H2O 0.05-0.015g; 0.5-1.5mL of bromothymol blue, 1000mL of distilled water, adjust the pH to 6.6-7.0; when the above culture medium is a liquid culture medium, the solid culture medium is added with 1-3% agar powder by mass on the basis of the above.

[0009] Preferably, the HNM medium includes HNM1 medium and HNM2 medium, and the DM medium includes DM1 medium and DM2 medium; the HNM1 medium formula is: (NH4)2SO4 0.014g, trisodium citrate 0.053g, K2HPO4·3H2O 0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl2 0.05g, distilled water 1000mL, adjusted to pH 7; the HNM2 medium formula is: (NH4)2SO4 0.47g, trisodium citrate 1.80g, K2HPO4·3H2O 0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl20.05g, distilled water 1000mL, adjusted to pH 7; the DM1 culture medium formula is: KNO30.72g, trisodium citrate 1.80g, K2HPO4·3H2O 0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl20.05g, distilled water 1000mL, adjusted to pH 7; the DM2 culture medium formula is: NaNO20.5g, trisodium citrate 1.80g, K2HPO4·3H2O 0.20g, Mg SO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl2 0.05g, distilled water 1000mL, adjust pH to 7.

[0010] The present invention also provides a method for quickly screening oligotrophic HN-AD bacteria using the culture medium, comprising the following steps: strain enrichment: mixing the mud sample with the HNM1 culture medium, and enriching it on a shaker at 28-32°C and 160-200rpm for 70-74h to obtain enriched culture solution 1; strain initial screening and preservation: taking the enriched culture solution 1 and mixing it with the HNM2 culture medium, and enriching it on a shaker at 28-32°C and 160-200rpm for 46-50h to obtain enriched solution 2; enriched culture solution 2 is concentrated according to 10 -1 -10 -8Perform concentration gradient dilution, mix well, take the diluted concentration solution and apply it on BTB solid culture medium, invert and culture at 26-30°C until a single colony grows; pick the single colonies with blue color around the colonies on the BTB solid plate and different shapes and sizes, streak and purify on the LB solid plate; pick the single colony on the streaked plate and inoculate it into HNM2 liquid culture medium, shake at 28-32°C, 160-200rpm for enrichment for 22-26h, obtain the bacterial solution, and preserve it; rapid rescreening: streak the bacterial solution on the LB plate, invert and culture at 26-30°C, take the single colony on the plate and inoculate it into LB liquid culture medium, shake at 28-32°C, 160-200rpm, after the strain is cultured, take the bacterial solution and centrifuge it, collect the precipitate, wash it, resuspend it, and prepare it into OD 600 0.8-1.2 bacterial suspension; inoculate the bacterial suspension into HNM2, DM1, and DM2 culture media respectively, set up blank controls, and culture at 160-200 rpm in a shaker at 28-32°C for 46-50 hours before testing. The target HN-AD strain with high-efficiency denitrification performance was screened out through the above three culture media.

[0011] Preferably, in the strain enrichment step, the mass volume ratio of the mud sample to the HNM1 culture medium is 3-7 g:180-220 mL.

[0012] Preferably, in the initial strain screening step, the volume ratio of the enrichment culture solution 1 to the HNM2 culture medium is 15-25 mL:80-120 mL.

[0013] Preferably, in the initial strain screening step, a solution of appropriate dilution concentration is applied to the BTB solid culture medium according to the growth of the strain.

[0014] Preferably, in the initial strain screening step, purification is performed 2-4 times using a four-zone streak method on an LB solid plate.

[0015] Preferably, the strain preservation step comprises: mixing 0.5-1.5 mL of bacterial solution with 0.3-0.7 mL of 50-70% glycerol and then freezing and storing the mixture at -75 to -85°C.

[0016] Preferably, in the rapid rescreening step, the bacterial suspension is inoculated into 180-220 μL of HNM2, DM1, and DM2 culture media at an inoculum volume of 1-3%.

[0017] Preferably, in the rapid rescreening step, the detection method includes: qualitatively detecting the NH4+-N concentration of the strain cultured with HNM2, and compared with the blank control group, the lighter the yellow color, the lower the NH4+-N concentration and the better the NH4+-N removal effect; qualitatively detecting the NO 2- -N concentration, compared with the blank control group, the lighter the red, the NO 2- The lower the -N concentration, the more NO2- -N effect is better; the growth of strains cultured in DM1 was evaluated, and the OD 600 Larger, NO 2- -N detection of lighter red is used as the selection criterion, indicating NO removal 3- -N is better and NO 2- -N accumulates less.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] First, the present invention utilizes low-nutrient (C / N=5) culture medium to enrich and separate the HN-AD strain, which is suitable for denitrification treatment of oligotrophic water bodies, and provides a low-carbon and safe biological treatment method for nitrogen removal in micro-polluted water bodies such as rivers, lakes and reservoirs.

[0020] Second, the screening method of the present invention greatly improves the screening efficiency. The traditional method takes 60-70 days to screen 100 strains of bacteria, while the present invention only takes 3-4 days; the traditional method only relies on the spectrophotometer to detect NH4 + -N, NO 2- -N and NO 3- The method of screening the samples based on the results of the -N concentration is complicated and will cause waste of human resources, reagents and consumables, etc. The present invention first uses different functional culture media to screen by observing the color with the naked eye, and then performs one three-state nitrogen concentration determination to complete the screening. The process is simple, the workload is small, and the efficiency can be improved by about 95%.

[0021] Third, the screening method of the present invention is carried out using a 96-well plate, which requires a small amount of sample and can screen hundreds or thousands of strains at the same time, making it very suitable for screening large-scale samples.

[0022] In summary, the rapid screening method of the present invention has strong selectivity, high accuracy, simplicity and rapidity, and is very suitable for screening large quantities of samples. It can also screen out oligotrophic HN-AD strains suitable for the treatment of nitrogen pollution in low-carbon and nitrogen water bodies, and may become a rapid screening method with significant application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of the rapid screening method for oligotrophic HN-AD bacteria.

[0024] Figure 2 After 48h of culture in HNM2 medium, NH4 + -N removes results.

[0025] Figure 3 DM1 medium was used to culture NO for 48h 2- -N removal results (the figure is a 200-fold dilution).

[0026] Figure 4 OD after 48h of culture in DM1 medium 600 result.

[0027] Figure 5 After 48 h of culture in DM2 medium, NO 2- -N Accumulate results.

[0028] Figure 6 is the screening verification result. Among them, a, NH4 + -N removal rate; b, NO 3- -N removal rate; c, OD at different time periods during HNM2 culture 600 ; d, OD at different time periods during DM1 culture 600 ; e, NO 3- -N removal process 48hNO 2- -The accumulation of N. DETAILED DESCRIPTION

[0029] The present invention provides a screening culture medium for oligotrophic HN-AD bacteria, including HNM culture medium, DM culture medium and BTB culture medium:

[0030] The HNM medium formula includes: (NH4)2SO4 0.014-0.47g, trisodium citrate 0.053-1.80g, K2HPO4·3H2O 0.10-0.30g, MgSO4·7H2O 0.03-0.07g, NaCl 0.08-0.16g, FeSO4·7H2O 0.005-0.015g, MnSO4·H2O 0.005-0.015g, CaCl2 0.03-0.07g, distilled water 1000mL, adjusted to pH 6.8-7.2; the DM medium formula includes: KNO3 0.5-0.72g, trisodium citrate 1.60-2.0g, K2HPO4·3H2O 0.10-0.30g, MgSO4·7H2O 0.03-0.07g, NaCl0.08-0.16g, FeSO4·7H2O 0.005-0.015g, MnSO4·H2O0.005-0.015g, CaCl20.03-0.07g, distilled water 1000mL, adjust the pH to 6.8-7.2; BTB medium: trisodium citrate 1.60-2.00g; NaNO30.51-0.71g; KH2PO40.50-1.50g; FeSO4·7H2O 0.10-0.30g; MnSO4·H2O0.05-0.015g; bromothymol blue 0.5-1.5mL, distilled water 1000mL, adjust the pH to 6.6-7.0.The HNM culture medium of the present invention comprises an HNM1 culture medium and an HNM2 culture medium, and the DM culture medium comprises a DM1 culture medium and a DM2 culture medium; preferably, the HNM1 culture medium comprises: (NH4)2SO4 0.014 g, trisodium citrate 0.053 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, and distilled water 1000 mL, and the pH is adjusted to 7; the HNM2 culture medium comprises: (NH4)2SO4 0.47 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl20.05g, distilled water 1000mL, adjusted to pH 7; the DM1 culture medium formula is: KNO30.72g, trisodium citrate 1.80g, K2HPO4·3H2O0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl20.05g, distilled water 1000mL, adjusted to pH 7; the DM2 culture medium formula is: NaNO20.5g, trisodium citrate 1.80g, K2HPO4·3H2O 0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl20.05g, distilled water 1000mL, adjust the pH to 7; when the above culture medium is a liquid culture medium, the solid culture medium is added with 1-3% agar powder by mass on the basis of the above.

[0031] The present invention also provides a method for quickly screening oligotrophic HN-AD bacteria using the culture medium, comprising the following steps:

[0032] (1) Strain enrichment: The mud sample was mixed with HNM1 medium and enriched in a shaker at 28-32°C and 160-200 rpm for 70-74 h to obtain enrichment culture fluid 1; (2) Strain screening and preservation: The enrichment culture fluid 1 was mixed with HNM2 medium and enriched in a shaker at 28-32°C and 160-200 rpm for 46-50 h to obtain enrichment culture fluid 2; the enrichment culture fluid 2 was concentrated to 10 -1 -10 -8Perform concentration gradient dilution, mix well, take the diluted concentration solution and spread it on BTB solid culture medium, invert and culture at 26-30°C until a single colony grows; pick up single colonies with blue color around the colonies on the BTB solid plate and of different shapes and sizes, and streak them on the LB solid plate for purification; pick up single colonies on the streaked plate and inoculate them into HNM2 liquid culture medium, shake at 28-32°C, 160-200rpm for enrichment for 22-26h, obtain bacterial solution, and preserve it; (3) Rapid rescreening: streak the bacterial solution on the LB plate, invert and culture at 26-30°C, take a single colony on the plate and inoculate it into LB liquid culture medium, shake at 28-32°C, 160-200rpm, after the strain is cultured, take the bacterial solution and centrifuge it, collect the precipitate, wash it, resuspend it, and prepare it into OD 600 0.8-1.2 bacterial suspension; inoculate the bacterial suspension into HNM2, DM1, and DM2 culture media respectively, set up blank controls, and culture them in a shaker at 28-32°C and 160-200rpm for 46-50h before testing. The target HN-AD strain with high denitrification performance was screened out through the above three culture media; (4) Quick screening verification: inoculate the target HN-AD strain bacterial suspension into HNM2 and DM1 liquid culture media, culture them in a shaker at 28-32°C and 160-200rpm, take samples at 0h, 12h, 24h, 36h, and 48h after culture, and measure OD 600 and NH4+-N, NO 2- -N and NO 3- -N concentration, and screened out the N8 strain with high denitrification performance.

[0033] In the present invention, in the strain enrichment step, the mass volume ratio of the mud sample to the HNM1 culture medium is 3-7g:180-220mL, preferably 5g:200mL. The present invention uses a Peterson mud sampler to collect mud samples from the bottom of the Macrobrachium rosenbergii breeding pond and puts them into a sampling bag for standby. The present invention needs to be sealed with a sterile film and then cultured in a shaking table. The temperature of the shaking table culture of the present invention is preferably 30°C, the rotation speed is 180rpm, and the time is 72h.

[0034] In the present invention, in the initial screening step of the strain, the volume ratio of the enriched culture solution to the HNM2 medium is 15-25mL:80-120mL, preferably 20mL:100mL. In the initial screening step of the strain of the present invention, according to the growth of the strain, a solution of appropriate dilution concentration is applied to the BTB solid medium; preferably 10 -6 -10 -8 The three gradient dilution concentration solutions are coated on the BTB solid culture medium. In the initial screening step of the strain of the present invention, the four-zone streak method is used on the LB solid plate for purification 2-4 times, preferably 3 times.

[0035] In the present invention, the strain preservation step includes: mixing 0.5-1.5 mL of bacterial solution with 0.3-0.7 mL of 50-70% glycerol and then freezing and storing at -75 to -85°C; preferably, mixing 1 mL of bacterial solution with 0.5 mL of 60% glycerol and then freezing and storing at -80°C.

[0036] In the present invention, in the rapid rescreening step, the bacterial suspension is inoculated into 180-220 μL HNM2, DM1, and DM2 culture media at 1-3% inoculation volume, preferably 2% inoculation volume. In the rapid rescreening step of the present invention, 2 mL of bacterial solution is centrifuged at 5000 rpm for 10 min.

[0037] In the present invention, in the rapid rescreening step, the detection method includes: qualitatively detecting the NH4+-N concentration of the strain cultured with HNM2, and compared with the blank control group, the lighter the yellow color, the lower the NH4+-N concentration, and the better the NH4+-N removal effect; qualitatively detecting NO 2- -N concentration, compared with the blank control group, the lighter the red, the NO 2- The lower the -N concentration, the more NO 2- -N effect is better; the growth of strains cultured in DM1 was evaluated, and the OD 600 Larger, NO 2- -N detection of lighter red is used as the selection criterion, indicating NO removal 3- -N works better.

[0038] In the present invention, in the rapid screening verification step, in order to exclude the influence of the strain itself, NH4+-N, NO 2- -N and NO3 - After sampling, the samples with -N concentration were centrifuged at 5000 rpm for 5 min, and the supernatant was taken for detection.

[0039] In the present invention, unless otherwise specified, all reagents or culture media are commercially available products well known to those skilled in the art.

[0040] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0041] Example 1

[0042] A screening medium and a rapid screening method for oligotrophic HN-AD bacteria, the flow diagram of the rapid screening method is as follows Figure 1 shown.

[0043] 1. Preparation of culture medium

[0044] HNM1 culture medium: (NH4)2SO4 0.014 g, trisodium citrate 0.053 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjust the pH to 7.

[0045] HNM2 culture medium: (NH4)2SO4 0.47 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjust the pH to 7.

[0046] DM1 culture medium: KNO3 0.72 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjust the pH to 7.

[0047] DM2 culture medium: NaNO2 0.5 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjust the pH to 7.

[0048] BTB medium: trisodium citrate 1.80 g, NaNO3 0.61 g, KH2PO4 1.00 g, FeSO4·7H2O 0.20 g, MnSO4·H2O 0.10 g, bromothymol blue 1 mL, distilled water 1000 mL, adjust the pH to 6.8.

[0049] LB medium: LB broth 20.00 g, distilled water 1000 mL.

[0050] When the above culture medium is a liquid culture medium, the solid culture medium is added with 2% agar powder (20.00 g) on ​​the basis of the above, and is sterilized in an autoclave at 121° C. for 30 min before use.

[0051] 2. Sample collection: Use Peterson mud sampler to collect mud samples from the bottom of the Macrobrachium rosenbergii breeding pond of Zhengda and put them into sampling bags for later use.

[0052] 3. Strain enrichment: The sterilized culture medium was dispensed into 200 mL to 250 mL conical flasks in a clean bench. 5 g of mud sample was weighed and placed into 200 mL of HNM1 culture medium. The mixture was placed in a constant temperature shaker at 30 °C and 180 rpm for enrichment for 72 h.

[0053] 4. Initial screening of strains: The sterilized culture medium was divided into 100 mL to 250 mL conical flasks in a clean bench. 20 mL of the above enrichment solution was added to 100 mL of HNM2 culture medium. After sealing with a sterile film, it was placed in a constant temperature shaker at 30°C and 180 rpm for enrichment for 48 h. The obtained enrichment culture solution was diluted with sterile water in a concentration gradient (10 -1 ~10 -8 ), vortexed and mixed, according to the growth of the strain, 10 -6 -10 -8 0.1 mL of three gradient dilution solutions were applied to BTB solid medium and inverted at 28°C until a single colony grew. Single colonies with different shapes and sizes with blue color around the colonies on the solid plate were picked and purified three times using the four-zone streak method on the LB solid plate. Single colonies on the streaked plate were picked and inoculated into a bacterial bottle containing 10 mL of HNM2 liquid medium, placed on a shaker, and cultured at 30°C and 180 rpm for 24 hours.

[0054] 5. Strain preservation (20% glycerol cryopreservation): prepare 60% glycerol: 60 mL glycerol + 40 mL pure water, sterilize in an autoclave at 121°C for 30 min (the bottle mouth should be loosened by half a circle to one circle during sterilization, and the cap should be quickly tightened when the autoclave is opened), add 0.5 mL 60% glycerol to the cryopreservation tube and then add 1 mL of bacterial solution to finally make 20% glycerol, label, and freeze at -80°C.

[0055] 6. Rapid rescreening: Take out the frozen strain and thaw it at 4℃, take 1μL of bacterial solution and streak it on the LB plate, incubate it at 28℃, take a single colony on the plate and inoculate it into a bacterial bottle containing 10mL LB liquid culture medium, put it in a constant temperature shaker, and culture it at 30℃ and 180rpm. After the strain is cultured, take 2mL of the bacterial solution, centrifuge it at 5000rpm for 10min, wash it twice with sterile water, resuspend it, and prepare it into OD 600 About 1.0 of bacterial suspension; the bacterial suspension was inoculated at a 2% inoculation rate into 96-well plates containing 200 μL HNM2, DM1, and DM2 culture media, with 6 parallels for each strain and 6 blank controls for each plate. The plates were cultured at 30°C in a shaker at 180 rpm for 48 hours before testing.

[0056] The detection method is as follows: (1) Qualitative detection of NH4 by strains cultured with HNM2+ -N concentration, compared with the blank control group, the lighter the color (yellow), the more NH4 + The lower the -N concentration, the more NH4 + -N has a better effect, and the result is as follows Figure 2 (2) The growth of the strain cultured in DM1 was evaluated. The turbidity and OD 600 Larger, NO 2- -N detection color (red) is lighter as the selection standard, indicating NO removal 3- -N has a better effect, and the result is as follows Figure 3 and Figure 4 (3) Qualitative detection of NO in strains cultured with DM2 2- -N concentration, compared with the blank control group, the lighter the color (red), the more NO 2- The lower the -N concentration, the more NO 2- -N has a better effect, and the result is as follows Figure 5 The target HN-AD strain with high denitrification performance was screened out by the above three culture media.

[0057] in Figure 2 In HMN2 medium, select the NH4 + The strains with better -N effects are N5, N6, N8, N34, N37, and N42; Figure 3 In DM1 medium, NO was selected according to the results. 2- -The strains with less N accumulation were N5, N8, N11, N14, N26, and N42; Figure 4 The strains that grew best in DM1 medium were N5, N6, N8, N14, N20, N42, and N37; Figure 5 In DM2 medium, select deNO according to the results 2- The strains with better -N effects are N8, N11, N20, N26, N34, and N37. According to the above results, strain N8 is a strain selected from the above three culture media, and N8 has high efficiency in denitrification.

[0058] 7. Rapid screening verification: Take out the bacterial solution stored in -80℃ and thaw it at 4℃. Use a pipette to draw 1μL of bacterial solution and streak it (HNM2 culture medium), then put it in a constant temperature incubator and invert it for culture at 28℃. Pick a single colony on the plate and inoculate it into a bacterial bottle containing 10mL of HNM2 culture medium, put it in a shaker, and culture it at 30℃ and 180rpm for 24h. Pipette 1mL of bacterial suspension and inoculate it into HNM2 and DM1 liquid culture medium (100mL), put it in a shaker, and culture it at 30℃ and 180rpm. Take samples at 0h, 12h, 24h, 36h, and 48h, and measure NH4 with 1mL. + -N, NO 2--N and NO 3- -N concentration; 3mL to measure OD 600 To exclude the influence of the strain itself, NH4 + -N, NO 2- -N and NO 3- -N concentration samples were collected and centrifuged at 5000rpm for 5 minutes, and the supernatant was taken for subsequent determination. The detection method was carried out according to Table 1. The determination results were as follows Figure 6 As shown, the strain N8 selected by the rapid screening and the strains that were not selected by the rapid screening method before (screened out) were selected to verify the accuracy of the rapid screening results. The final results verified that the N8 strain had a better denitrification effect and less nitrite accumulation, and the verification results were consistent with the above rapid screening results.

[0059] Table 1 Detection methods of various detection indicators

[0060] Detection indicators Detection Methods <![CDATA[NH4 + -N]]> Nessler's reagent method, HJ535-2009 <![CDATA[NO3 - -N]]> UV spectrophotometer method, HJ / T346-2007 <![CDATA[NO2 - -N]]> Spectrophotometer method, GB7493-87 <![CDATA[OD 600 ]]> Spectrophotometer

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A screening medium for oligotrophic HN-AD bacteria, characterized in that: Including HNM medium, DM medium and BTB medium: The HNM medium formula includes: (NH4)2SO4 0.014-0.47 g, trisodium citrate 0.053-1.80 g, K2HPO4·3H2O 0.10-0.30 g, MgSO4·7H2O 0.03-0.07 g, NaCl 0.08-0.16 g, FeSO4·7H2O 0.005-0.015 g, MnSO4·H2O 0.005-0.015 g, CaCl2 0.03-0.07 g, distilled water 1000 mL, adjusted to pH 6.8-7.2; The DM medium formula includes: KNO3 0.5-0.72 g, trisodium citrate 1.60-2.0 g, K2HPO4·3H2O 0.10-0.30 g, MgSO4·7H2O 0.03-0.07 g, NaCl 0.08-0.16 g, FeSO4·7H2O 0.005-0.015 g, MnSO4·H2O 0.005-0.015 g, CaCl2 0.03-0.07 g, distilled water 1000 mL, adjusted to pH 6.8-7.2; The BTB culture medium formula includes: trisodium citrate 1.60-2.00 g; NaNO3 0.51-0.71 g; KH2PO4 0.50-1.50 g; FeSO4·7H2O 0.10-0.30 g; MnSO4·H2O 0.05-0.015 g; bromothymol blue 0.5-1.5 mL, distilled water 1000 mL, adjusted to pH 6.6-7.

0. When the above culture medium is a liquid culture medium, the solid culture medium is added with 1-3% agar powder by mass on the basis of the above.

2. The screening medium according to claim 1, characterized in that The HNM culture medium includes HNM1 culture medium and HNM2 culture medium, and the DM culture medium includes DM1 culture medium and DM2 culture medium; The HNM1 culture medium formula is: (NH4)2SO4 0.014g, trisodium citrate 0.053g, K2HPO4·3H2O 0.20g, MgSO4·7H2O 0.05g, NaCl 0.12g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g, CaCl2 0.05g, distilled water 1000mL, adjusted to pH 7; The HNM2 culture medium formula is: (NH4)2SO4 0.47 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjusted to pH 7; The DM1 culture medium formula is: KNO3 0.72 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, adjusted to pH 7; The DM2 culture medium formula is: NaNO2 0.5 g, trisodium citrate 1.80 g, K2HPO4·3H2O 0.20 g, MgSO4·7H2O 0.05 g, NaCl 0.12 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, CaCl2 0.05 g, distilled water 1000 mL, and the pH is adjusted to 7.

3. A method for rapidly screening oligotrophic HN-AD bacteria using the culture medium according to claim 1 or 2, characterized in that: The steps include: Strain enrichment: Mix the mud sample with HNM1 medium and shake it at 28-32°C and 160-200 rpm for 70-74 hours to obtain enrichment culture solution 1; Initial screening and preservation of strains: enrichment culture fluid 1 was mixed with HNM2 medium, and the mixture was shaken at 28-32°C and 160-200 rpm for 46-50 h to obtain enrichment culture fluid 2; enrichment culture fluid 2 was added according to 10 -1 -10 -8 Perform concentration gradient dilution, mix well, take the diluted concentration solution and apply it on BTB solid culture medium, invert and culture at 26-30°C until a single colony grows; pick single colonies with blue color around the colonies on the BTB solid plate and different shapes and sizes, streak and purify on the LB solid plate; pick single colonies on the streaked plate and inoculate them into HNM2 liquid culture medium, shake at 28-32°C, 160-200rpm for enrichment for 22-26h, obtain bacterial solution, and preserve; Rapid rescreening: Streak the bacterial solution on an LB solid plate, invert and culture at 26-30°C, take a single colony from the plate and inoculate it into LB liquid culture medium, culture it on a shaker at 28-32°C and 160-200rpm. After the strain is cultured, centrifuge the bacterial solution, collect the precipitate, wash it, resuspend it, and prepare it into OD 600 0.8-1.2 bacterial suspension; inoculate the bacterial suspension into HNM2, DM1, and DM2 culture media respectively, set up blank controls, and culture at 160-200 rpm in a shaker at 28-32°C for 46-50 hours before testing. The target HN-AD strain with high-efficiency denitrification performance was screened out through the above three culture media.

4. The method according to claim 3, characterized in that In the strain enrichment step, the mass volume ratio of the mud sample to the HNM1 culture medium is 3-7 g:180-220 mL.

5. The method according to claim 3, characterized in that In the initial strain screening step, the volume ratio of the enrichment culture solution 1 to the HNM2 culture medium is 15-25 mL:80-120 mL.

6. The method according to claim 3, characterized in that In the initial strain screening step, according to the growth of the strain, a solution of appropriate dilution concentration was applied to the BTB solid culture medium.

7. The method according to claim 3, characterized in that In the initial strain screening step, the four-zone streak method was used on LB solid plates for purification 2-4 times.

8. The method according to claim 3, characterized in that The strain preservation step includes: mixing 0.5-1.5 mL of bacterial solution with 0.3-0.7 mL of 50-70% glycerol and then freezing and storing at -75 to -85°C.

9. The method according to claim 3, characterized in that In the rapid rescreening step, the bacterial suspension was inoculated into 180-220 μL HNM2, DM1, and DM2 culture media at a 1-3% inoculum volume.

10. The method according to claim 3, characterized in that In the rapid rescreening step, the detection methods include: qualitative detection of NH4+-N concentration of strains cultured with HNM2. Compared with the blank control group, the lighter the yellow, the lower the NH4+-N concentration and the better the NH4+-N removal effect; qualitative detection of NO by strains cultured with DM2 2- -N concentration, compared with the blank control group, the lighter the red, the NO 2- The lower the -N concentration, the more NO 2- -N effect is better; the growth of strains cultured in DM1 was evaluated, and the OD 600 Larger, NO 2- -N detection of lighter red is used as the selection criterion, indicating NO removal 3- -N is better and NO 2- -N accumulates less.

Images