CN107446840B - 2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium and application thereof - Google Patents
2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium and application thereof Download PDFInfo
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- CN107446840B CN107446840B CN201710556797.7A CN201710556797A CN107446840B CN 107446840 B CN107446840 B CN 107446840B CN 201710556797 A CN201710556797 A CN 201710556797A CN 107446840 B CN107446840 B CN 107446840B
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
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- C—CHEMISTRY; METALLURGY
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention provides a degrading bacterium of 2-phenyl-6-hydroxy-3-pyridazinone, which is identified as bacillus aerophilusBacillus aerophilusThe compound microbial inoculum belongs to gram-positive bacteria, which are preserved in China general microbiological culture Collection center (CGMCC) in 26.5.2017, the preservation number is CGMCC NO.14198, 2-phenyl-6-hydroxy-3-pyridazinone can be used as a unique carbon source and energy source for growth, the degradation rate of 100 mg/L2 of 2-phenyl-6-hydroxy-3-pyridazinone in 32 hours reaches over 90 percent, the problem that the 2-phenyl-6-hydroxy-3-pyridazinone generated in the pesticide production process is difficult to degrade is solved, the pesticide production wastewater can reach the standard and be discharged, and the important effects on the clean production and environmental protection of the 2-phenyl-6-hydroxy-3-pyridazinone are achieved.
Description
Technical Field
The invention belongs to the technical field of biodegradation bacteria, and particularly relates to 2-phenyl-6-hydroxy-3-pyridazinone degradation bacteria and application thereof.
Background
2-phenyl-6-hydroxy-3-pyridazinone is an intermediate of organophosphorus insecticide pyridaphenthion, and has a unique heterocyclic structure, so that waste water generated in the production process is not effectively treated so far, the problem of serious environmental pollution is also caused, and the restoration of the polluted environment draws wide attention.
The microbial degradation has the advantages of low cost, high efficiency, no secondary pollution, good ecological restoration and the like, and is applied to many aspects, so that the screening of the 2-phenyl-6-hydroxy-3-pyridazinone degrading bacteria has great significance for treating 2-phenyl-6-hydroxy-3-pyridazinone wastewater.
Disclosure of Invention
The invention aims to provide a 2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium and application thereof, and the bacterium agent prepared by the bacterium can enable the degradation rate of the 2-phenyl-6-hydroxy-3-pyridazinone to reach more than 90 percent, and has lower production and use cost.
The invention provides a 2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium, wherein a strain NZJ-52 is a gram-positive bacterium, and has the main biological characteristics of rod-shaped thallus, white and opaque bacterial colony, incomplete edge, mesogenic spore with the diameter of 1-2mm, optimal growth temperature of 30 ℃ and pH of 7.0. It has been deposited in China general microbiological culture Collection center (CGMCC) at 27.5.2017, with the following addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.14198, identified as Bacillus aerophilusBacillus aerophilusThe 2-phenyl-6-hydroxy-3-pyridazinone can be used as a unique carbon source and a unique nitrogen source for growth, and the growth is mineralized to release ammonia nitrogen.
The invention provides a microbial inoculum of the bacillus aerophilus NZJ-52.
The invention also provides a method for producing a microbial inoculum by the bacillus aerophilus NZJ-52.
The packaging dosage form of the product is a liquid microbial inoculum or a solid adsorption microbial inoculum.
The method for producing the microbial inoculum comprises the following specific steps:
1) inoculating a test tube seed of 2-phenyl-6-hydroxy-3-pyridazinone degrading bacteria bacillus aerophilus NZJ-52 into L B culture medium, and carrying out shake culture until logarithmic phase, wherein the formula of the L B culture medium comprises 10.00 g/L of NaCl, 10.00 g/L of peptone, 5.00 g/L of yeast powder and pH 7.0;
2) inoculating the cultured strain in step 1) into 500L seed tank according to 10% of inoculum size, culturing to logarithmic phase, wherein the seed tank contains culture medium formula of 0.8% glucose (NH)4）2SO41%，K2HPO40.2%，MgSO40.05%，NaCl0.01%，CaCO30.3 percent, 0.02 percent of yeast extract and 7.2 to 7.5 of pH value.
3) Inoculating the seed solution obtained in the step 2) into a production tank according to the inoculation amount of 10% for fermentation culture, wherein the culture medium used by the production tank is the same as that of the seed tank, and the culture solution after fermentation is the microbial inoculum.
In the culture process of the seeding tank in the step 2) and the production tank in the step 3), the ventilation volume of sterile air is 1: 0.6 to 1.2, the stirring speed is 180-240r/min, the culture temperature is 30 to 35 ℃, and the culture time of the whole process flow of the steps 2 and 3 is 48 to 60 hours.
The microbial inoculum is applied to the treatment of the production wastewater of organophosphorus insecticide pyridaphenthion, and the degradation object is 2-phenyl-6-hydroxy-3-pyridazinone.
Has the advantages that:
the bacillus aerophilus NZJ-52 has the degradation rate of more than 90 percent on the 2-phenyl-6-hydroxy-3-pyridazinone, greatly reduces the workload in the production and use processes, and reduces the production and use cost, the invention has important significance for protecting the ecological environment and reducing the cost of wastewater treatment, the prepared microbial inoculum has the advantages of low production and use cost, convenient use and good removal effect, the degrading bacteria NZJ-52 can grow on a culture medium taking the 2-phenyl-6-hydroxy-3-pyridazinone as the only carbon source, the degradation rate of 100 mg/L2 of the 2-phenyl-6-hydroxy-3-pyridazinone reaches more than 90 percent according to 1 percent of inoculation amount in an inorganic salt culture medium containing the 2-phenyl-6-hydroxy-3-pyridazinone, the degradation rate of the 2-phenyl-6-hydroxy-3-pyridazinone reaches more than 90 percent under the conditions of 30 ℃ and pH7.0, the problem of degrading the 2-phenyl-6-hydroxy-3-pyridazinone in the oscillation culture process for 32 hours is solved, and the 2-phenyl-6-hydroxy-3-pyridazinone produced in the production process has the important effect of protecting the wastewater and ensuring the emission to reach the standard.
Drawings
FIG. 1 is a colony morphology diagram of Bacillus aerophilus NZJ-52;
FIG. 2 Shake flask degradation plot of B.aerophilus NZJ-52.
Detailed Description
Example 1 isolation and characterization of the strains
2-phenyl-6-hydroxy-3-pyridazinone is prepared into 10-1The enrichment solution is sucked by 1.0 mlPrepared 10-1Adding the enriched solution into 9.0 ml of sterile water, and fully and uniformly mixing to prepare 10-2The enrichment solution is diluted in a gradient manner, 0.1 ml of each gradient of the dilution solution is absorbed and coated on an inorganic salt solid culture medium (the formula is that each liter contains 1.50 g K) with the concentration of 100 mg/L and containing 2-phenyl-6-hydroxy-3-pyridazinone2HPO4、0.50 g KH2PO4、0.20 g MgSO4×7H2O、1.00 gNaCl、1.00 g (NH4)2SO420.00 g agar, pH 7.0), and cultured at 30 ℃ for 7 days. And selecting a single colony from the bacterial strains to verify the degradation effect, storing a strain with higher degradation efficiency, and performing subsequent experiments. The colony pattern is shown in FIG. 1 and identified as Bacillus aerophilusBacillus aerophilus. The main biological characteristics are that the bacterial colony is white and non-transparent, the edge is incomplete, the diameter is 1-2mm, the optimum growth temperature is 30 ℃, and the pH value is 7.0. Can grow by taking 2-phenyl-6-hydroxy-3-pyridazinone as a unique carbon source and a unique nitrogen source, and mineralizes the carbon source and the nitrogen source to generate ammonia nitrogen.
Example 2 Shake flask degradation experiment of Bacillus aerophilus NZJ-52
In an inorganic salt medium containing 100 mg/L of 2-phenyl-6-hydroxy-3-pyridazinone, the formula comprises 1.50 g 1.50 g K2HPO4, 0.50g KH2PO4, 0.20g MgSO4 × 7H2O, 1.00g NaCl, 1.00g (NH4)2SO4 and pH7.0 per liter, NZJ-52 is inoculated according to the inoculation amount of 1 percent, the culture is shaken at 30 ℃ and samples are taken at intervals of 4 hours for determination, and as can be seen from figure 2, the 2-phenyl-6-hydroxy-3-pyridazinone is degraded by more than 90 percent within 32 hours.
Example 3 preparation of a Bacillus aerophilus NZJ-52-containing preparation
Activating the aerophilic bacillus NZJ-52 on a culture dish, inoculating the activated aerophilic bacillus NZJ-52 on a test tube inclined plane (a culture medium formula: NaCl10.00 g/L, peptone 10.00 g/L, yeast powder 5.00 g/L, 2% agar, pH 7.0) for standby, inoculating a test tube seed into a 1000ml shake flask containing 200ml L B culture medium, wherein the formulation of the L B culture medium is 5.00 g/L of yeast extract, 10.00 g/L/L, pH7.0, carrying out constant temperature shaking culture to a logarithmic phase, preparing to inoculate a seed tank, and the seed tank is 500 liters, the feeding amount is 400 liters, the culture medium formula is 0.8% of glucose (NH)4）2SO41%，K2HPO40.2%，MgSO40.05%，NaCl 0.01%， CaCO30.3 percent, 0.02 percent of yeast extract and 7.2 to 7.5 of pH value. After the feeding is finished, high-pressure moist heat sterilization is carried out at 121 ℃, after the temperature is cooled to 33 ℃, the cultured shake flask strain is inoculated into a 500-liter seeding tank according to the inoculation amount of 10 percent, the strain is cultured to the logarithmic phase, the stirring speed is 220 r/min, and the introduction amount of sterile air is 1: 0.8. Inoculating the seed liquid reaching logarithmic phase into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium composition of the production tank is the same as that of a seed tank. The capacity of the production tank is 5 tons, and the feeding amount is 4.5 tons. 1.1kg/cm production tank after feeding2Sterilizing at 121 deg.C, cooling to below 35 deg.C, and introducing sterile air to maintain sterile state. The temperature of the production tank after inoculation is controlled at 35 ℃, the ventilation quantity of sterile air in the culture process of the production tank is 1: 1.2, the stirring speed is 240r/min, and the culture time of the whole process flow is 60 hours. The number of the thalli after fermentation is over 10 hundred million/ml. After fermentation, the culture solution is directly taken out of the tank and is subpackaged into liquid dosage forms by using a plastic packaging barrel or a packaging bottle or into solid microbial inoculum dosage forms by adopting a packaging bag for peat adsorption.
Example 4 application of Bacillus aerophilus NZJ-52 in treatment of production wastewater of organophosphorus insecticide pyridaphenthion
1 ton of wastewater of an organophosphorus pesticide production plant is taken, primary treatment is carried out, solid suspended matters in the wastewater are removed by using a grating, a screen and a sedimentation tank, the content of 2-phenyl-6-hydroxy-3-pyridazinone in the initial wastewater is 5820 mg/L, 10 kg of solid microbial inoculum is added after the pH value is primarily adjusted, the content of 2-phenyl-6-hydroxy-3-pyridazinone is 232.8 mg/L after 32 hours, and the degradation rate of the benzene thiazole alcohol is found to reach 96%.
Claims (7)
1. A2-phenyl-6-hydroxy-3-pyridazinone degrading bacterium is named as bacillus aerophilusBacillus aerophilus) NZJ-52, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC NO. 14198.
2. A microbial agent comprising the 2-phenyl-6-hydroxy-3-pyridazinone-degrading bacterium according to claim 1.
3. The method for preparing the microbial inoculum according to claim 2, which comprises the following steps:
step 1), inoculating test tube strains of 2-phenyl-6-hydroxy-3-pyridazinone degrading bacteria into L B culture medium, and carrying out shake culture until logarithmic phase;
step 2), inoculating the cultured strains into a seeding tank according to the inoculation amount of 10 percent, and culturing to logarithmic phase;
step 3), inoculating the seed solution into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium used by the production tank is the same as the culture medium used by the seeding tank;
the ventilation quantity of sterile air in the culture process of the seeding tank and the production tank is 1: 0.6-1.2, the stirring speed is 180-240r/min, the culture temperature is 30-35 ℃, the culture time of the whole process flow is 48-60 h, the number of thalli reaches more than 10 hundred million/m L after the fermentation is finished, and the culture solution is directly subpackaged into liquid microbial inoculum from the tank or solid microbial inoculum from a packaging bag for peat adsorption after the fermentation is finished.
4. The method for preparing microbial inoculum according to claim 3, wherein the L B culture medium formula comprises NaCl10.00 g/L, peptone 10.00 g/L, yeast powder 5.00 g/L, and pH 7.0.
5. The method for preparing the microbial inoculum according to claim 3, wherein the formula of the culture medium used by the seeding tank is as follows: glucose 0.8%, (NH)4）2SO41%，K2HPO40.2%，MgSO40.05%，NaCl 0.01%，CaCO30.3 percent, 0.02 percent of yeast extract and 7.2 to 7.5 of pH value.
6. The use of the 2-phenyl-6-hydroxy-3-pyridazinone degrading bacteria of claim 1 in the treatment of production wastewater of organophosphorus insecticide pyridaphenthion.
7. Use of the 2-phenyl-6-hydroxy-3-pyridazinone-degrading bacterium according to claim 1 or the microbial agent according to claim 2 for degrading 2-phenyl-6-hydroxy-3-pyridazinone.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302865A (en) * | 2001-01-17 | 2001-07-11 | 南京农业大学 | Bacterium for degradating residual agricultural organophosphorus chemical and its bacterial preparation |
| CN103952353A (en) * | 2014-04-24 | 2014-07-30 | 烟台海上传奇生物科技有限公司 | Bacillus aerophilus, microbial agent and applications of bacillus aerophilus and microbial agent |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302865A (en) * | 2001-01-17 | 2001-07-11 | 南京农业大学 | Bacterium for degradating residual agricultural organophosphorus chemical and its bacterial preparation |
| CN103952353A (en) * | 2014-04-24 | 2014-07-30 | 烟台海上传奇生物科技有限公司 | Bacillus aerophilus, microbial agent and applications of bacillus aerophilus and microbial agent |
Non-Patent Citations (2)
| Title |
|---|
| Biodegradation of imidacloprid in sandy loam soil by Bacillus aerophilus;Romila Akoijam et al.;《Intern. J. Environ. Anal. Chem.》;20150612;第95卷(第8期);第730-743页 * |
| 菌株 scl-2 对苯线磷、哒嗪硫磷和丙溴磷降解的研究;李荣等;《中国环境科学》;20111231;第31卷(第7期);第1178-1185页 * |
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