CN106554921B - Preservation method of nitrifying bacteria - Google Patents
Preservation method of nitrifying bacteria Download PDFInfo
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- CN106554921B CN106554921B CN201510635129.4A CN201510635129A CN106554921B CN 106554921 B CN106554921 B CN 106554921B CN 201510635129 A CN201510635129 A CN 201510635129A CN 106554921 B CN106554921 B CN 106554921B
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Abstract
The invention discloses a method for preserving nitrobacteria, which comprises the following steps of (1) adding a growth promoter in the culture process of the nitrobacteria, wherein the growth promoter comprises metal salt and polyamine substances, the metal salt accounts for 40-100 parts by weight, and the polyamine substances account for 5-30 parts by weight; the metal salt consists of calcium salt, copper salt, magnesium salt and/or ferrous salt; after the culture reaches the growth stabilization period, collecting thalli; (2) preparing a nitrifying bacteria preservation nutrient solution; (3) mixing the nitrifying bacteria collected in the step (1) with the preservation nutrient solution prepared in the step (2), and controlling the water content to be 40-80%; (4) adding a preservative; (5) and (5) low-temperature freezing preservation. The growth promoter is added in the process of culturing the nitrobacteria, so that the thalli can be rapidly proliferated, the culture time is shortened, particularly, the activity and low temperature resistance of the thalli can be improved, and the activity can be rapidly recovered after low-temperature freezing storage.
Description
Technical Field
The invention relates to a method for preserving bacteria, in particular to a method for preserving nitrobacteria by low-temperature freezing.
Background
The strain is one of the main biological resources, and after a good strain is selected and bred, the good property of the good strain must be kept unchanged or slowly changed as little as possible, so that the performance of the strain is not reduced, and the good strain can be applied to production for a long time. The strain preservation method has a plurality of methods, the cryopreservation is an effective way for solving the germplasm degradation and preventing the naturally accumulated mutation, but the traditional cryopreservation needs expensive program cooling instruments and has complicated steps. Complete vitrification is a new method for low-temperature preservation, namely, under the condition of high-concentration protective agent, the cells and the protective agent are all in a vitrification state in the process of rapid cooling, the formation of ice crystals in cells is avoided, and all parts of organs and tissues are all in the same state.
The basic principle of microbial strain preservation is that conditions are artificially created according to the physiological and biochemical characteristics of microbes, so that the metabolism of the microbes is in a dormant state in which the microbes are inactive and the growth and the reproduction are inhibited, namely, the strains are temporarily in the dormant state by adopting the conditions of low temperature, drying, oxygen deficiency and the like. A good preservation method firstly can keep the original excellent characters of the strains unchanged for a long time, and simultaneously needs to consider the simplicity and the economy of the method so as to be popularized and used in production. Therefore, it is important to improve the low temperature resistance of the cells and to maintain the excellent performance of the cells and to prevent the degradation of the bacterial species, on the premise of determining an appropriate preservation method.
CN201110353746.7 discloses a nitrifying bacteria preservation method, which comprises the following steps: (1) culturing the nitrifying bacteria to a growth stabilization phase, and collecting the nitrifying bacteria; (2) preparing a nitrifying bacteria preservation nutrient solution; (3) mixing the nitrifying bacteria collected in the step (1) with the nitrifying bacteria preservation nutrient solution prepared in the step (2), wherein the water content is 40-80%, and the water content refers to the ratio of the wet weight of the bacteria to the volume of the culture solution; (4) adding a preservative; (5) and (5) freezing and preserving. The method can prolong the storage time of the strain, and simultaneously keep the activity of the strain relatively stable. However, the nitrifying bacteria grow slowly, and therefore the period required for culturing the strain to the stationary phase is long. In addition, in the process of low-temperature preservation, high-concentration nutrient substances and a large amount of preservative agents are required to be added, so that the preservation cost is high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preservation method of nitrifying bacteria. The growth promoter is added in the process of culturing the nitrobacteria, so that the thalli can be rapidly proliferated, the culture time is shortened, particularly, the activity and the low temperature resistance of the thalli can be improved, the activity can be rapidly recovered after the thalli are frozen and stored at a low temperature, and the storage cost is lower.
The preservation method of nitrifying bacteria comprises the following steps:
(1) adding a growth promoter in the process of culturing nitrobacteria, wherein the growth promoter comprises 40-100 parts by weight of metal salt and preferably 50-80 parts by weight of polyamine substance, and the polyamine substance is 5-30 parts by weight and preferably 10-20 parts by weight; the metal salt consists of calcium salt, copper salt, magnesium salt and/or ferrous salt; after the culture reaches the growth stabilization period, collecting thalli;
(2) preparing a nitrifying bacteria preservation nutrient solution;
(3) mixing the nitrifying bacteria collected in the step (1) with the preservation nutrient solution prepared in the step (2), and controlling the water content to be 40% -80%, preferably 50% -70%;
(4) adding a preservative;
(5) and (5) low-temperature freezing preservation.
The culture of the nitrifying bacteria in step (1) of the present invention is performed by a method conventional in the art, such as a batch-type activated sludge process. The culture solution can be artificially prepared, and the nitrifying bacteria can also be cultured by adopting waste water containing ammonia nitrogen, wherein the components contained in the culture solution are mainly ammonium salt, and the ammonia nitrogen concentration is 200-1500 mg/L. The initial source of nitrifying bacteria culture may be any nitrifying bacteria that require preservation. The culture conditions of the nitrifying bacteria are as follows: the pH value is 7.0-8.5, preferably 7.5-8.0; the temperature is 20-40 ℃, preferably 25-37 ℃; the dissolved oxygen concentration is 1-5mg/L, preferably 2-4 mg/L. Culturing the nitrifying bacteria until the nitrifying bacteria just enter a stable period, and collecting the obtained nitrifying bacteria by methods of sedimentation, filtration, centrifugation and the like.
The metal salt in the growth promoter of step (1) of the present invention may be calcium salt, magnesium salt and copper salt, wherein Ca is2+、Mg2+And Cu2+The molar ratio of (5-15): (5-25): 0.5-5), preferably (8-12): 10-20): 1-4; or salts of calcium, ferrous and copper, where Ca2+、Fe2+And Cu2+The molar ratio of (5-15): (1-8): 0.5-5, preferably (8-12): 2-6): 1-4; or calcium, magnesium, ferrous and copper salts, of which Ca is present2+、Mg2+、Fe2+And Cu2+The molar ratio of (5-15): (5-25)(1-8): (0.5-5), preferably (8-12): (10-20): 2-6): 1-4).
The calcium salt in the growth promoter in the step (1) is CaSO4Or CaCl2Preferably CaSO4(ii) a The magnesium salt is MgSO4Or MgCl2Preferably MgSO (MgSO)4(ii) a The ferrous salt is FeSO4Or FeCl2Preferably FeSO4(ii) a The copper salt being CuSO4Or CuCl2Preferably CuSO4。
The polyamine substance in the growth promoter in the step (1) of the invention is spermine, spermidine or a mixture of spermine and spermidine. Preferably, a certain amount of inorganic hydroxylamine acid, such as one or more of hydroxylamine hydrochloride, hydroxylamine sulfate or hydroxylamine phosphate, is added into the growth promoter, and the inorganic hydroxylamine acid is preferably hydroxylamine sulfate; the amount added is 0.5 to 15 parts by weight, preferably 2 to 10 parts by weight. The appropriate amount of the inorganic hydroxylamine acid can be used as a matrix of hydroxylamine oxidoreductase to directly participate in the metabolic process of nitrobacteria, shorten the enzymatic reaction process, and simultaneously can be used as an activator of cells to accelerate the growth of the cells.
The growth promoter in the step (1) is supplemented according to the concentration of the promoter in the culture system of 10-50mg/L, preferably 20-30 mg/L.
The ammonium salt contained in the nitrifying bacteria preserving nutrient solution in the step (2) of the invention is (NH)4)2SO4And also contains trace substance FeSO4、KH2PO4、MgCl2And CaCl2. The amount of each substance used is determined according to the concentration required in step (3). In the mixture of the nitrifying bacteria and the preserved nutrient solution, NH4 +-N concentration of 0.1-1.5g/L, Fe2+The concentration is 0.01-0.03g/L, K+Ca with a concentration of 0.05-0.5g/L2+The concentration is 0.01-0.05g/L, Mg2+The concentration is 0.05-0.25 g/L. The water content of the bacterial suspension has influence on the formation of ice crystals inside and outside cells and further on the preservation effect, so that the proper water content is required, the water content of the preservation system is controlled to be 40-80%, preferably 50-70%, and the water content refers to the weight content of water in the nitrite bacteria preservation system。
The preservative in step (4) of the present invention may be any conventional preservative used in the art, preferably dimethyl sulfoxide, and is used in an amount of 0.5 to 3% by volume of the mixture of nitrifying bacteria and preserving nutrient solution. Dimethyl sulfoxide is a permeability protective agent, has no toxicity to cells, small molecular weight and good solubility, can quickly penetrate into cells, reduce the freezing point, improve the permeability of cell membranes to water, delay the freezing process, enable water in the cells to penetrate out of the cells before freezing, form ice crystals outside the cells, improve the electrolyte concentration in the cells, reduce the ice crystals in the cells and further reduce the frostbite of the ice crystals on the cells.
In the step (5), the low-temperature freezing preservation temperature is-20 ℃ to-70 ℃. Refrigeration equipment conventional in the art may be employed.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, a specific growth promoter is added in the process of culturing the nitrobacteria, so that the thalli can be rapidly proliferated under the combined action of metal salt, polyamine substances and the like, the culture time is shortened, and the problem of slow growth of the nitrobacteria is solved.
2. The addition of the growth promoter can provide a good nutritional environment for the thallus cells, improve the stability and the freezing resistance of the preserved thallus and prolong the preservation time of the strain. The thallus stored by low-temperature freezing can quickly recover activity in a short time, and has high survival rate and strong impact resistance.
3. The growth promoter is added during the culture of the nitrobacteria, and the metal salt in the growth promoter not only is a nutrient solution, but also plays a role of a protective agent buffer solution, so that the use amount of a preservative agent is reduced, and the activity inhibition on the strains is reduced.
Detailed Description
The present invention will be described in detail with reference to examples. But are not to be construed as limiting the invention.
Firstly, preparing metal salt solution according to the proportion and the formula of the growth promoter shown in the table 1, and adding polyamine substances and inorganic hydroxylamine acid into the metal salt solution before use to prepare the growth promoters I-V with different models, wherein the concentration of the growth promoters is 0.5 g/L.
TABLE 1 formulation and proportions of the different accelerators
Example 1
Preparing a culture solution with the ammonia nitrogen concentration of 300mg/L in the inlet water, and culturing nitrobacteria in a 100L biological aeration reaction tank by adopting an intermittent activated sludge method, wherein the temperature is 27 ℃, the pH value is 7.8, and the dissolved oxygen DO is 2.5-3.5 mg/L. The growth promoter I was added in an amount of 25mg/L of the promoter concentration in the culture system. And stopping culturing after the thalli enters a stable growth period, wherein the culturing time is 3 days, washing for many times after sedimentation or centrifugation, and collecting the thalli. Then mixing the thallus with the prepared preservation nutrient solution according to the water content of 50 percent, shaking up, adding 1.5 percent of preservative dimethyl sulfoxide, and preserving in an ultra-low temperature refrigerator at 50 ℃ below zero. After 3 years, the activity and the survival rate of the thalli are taken out and examined, and after 2d recovery, the activity of the thalli can be completely recovered, the survival rate can reach 95 percent, and the ammonia nitrogen removal rate can reach more than 98 percent.
The concentrations of the nutrients in the final mixture were: 0.3g/L NH4 +-N, 0.01g/L Fe2+0.05g/L of Mg2+0.1g/L of K+0.01g/L of Ca2+。
Example 2
Preparing a culture solution with the ammonia nitrogen concentration of inlet water of 400mg/L, and culturing nitrobacteria in a 100L biological aeration reaction tank by adopting an intermittent activated sludge method, wherein the temperature is 37 ℃, the pH value is 7.8, and the dissolved oxygen DO is 0.5-1.5 mg/L. Adding growth promoter II according to the concentration of promoter in culture system of 30 mg/L. And stopping culturing after the thalli enters a stable growth period, wherein the culturing time is 4 days, washing for many times after sedimentation or centrifugation, and collecting the thalli. Then mixing the thallus with the prepared nutrient solution according to the water content of 70%, shaking up, adding 2.0% of preservative dimethyl sulfoxide, and preserving in an ultra-low temperature refrigerator at-70 ℃. After 5 years, the activity and the survival rate of the thalli are taken out and examined, and the activity of the thalli can be completely recovered after 4d recovery, the survival rate can reach 90 percent, and the ammonia nitrogen removal rate can reach more than 94 percent.
The concentrations of the nutrients in the final mixture were: 0.4g/L NH4 +-N, 0.01g/L Fe2+0.05g/L of Mg2+0.1g/L of K+0.01g/L of Ca2+。
Example 3
Preparing a culture solution with the inlet water ammonia nitrogen concentration of 600mg/L, and culturing nitrobacteria in a 100L biological aeration reaction tank by adopting an intermittent activated sludge method, wherein the temperature is 27 ℃, the pH value is 7.8, and the dissolved oxygen DO is 2.5-3.5 mg/L. The growth promoter III was added in accordance with the promoter concentration of 20mg/L in the culture system. And stopping culturing after the thalli enters a stable growth period, wherein the culturing time is 7 days, washing for multiple times after sedimentation or centrifugation, and collecting the thalli. Then mixing the thallus with the prepared nutrient solution according to the water content of 60%, shaking up, adding 2.5% of preservative dimethyl sulfoxide, and preserving in an ultra-low temperature refrigerator at-60 ℃. After 4 years, the activity and the survival rate of the thalli are taken out and examined, and after 3d recovery, the activity of the thalli can be completely recovered, the survival rate can reach 92 percent, and the ammonia nitrogen removal rate can reach more than 95 percent.
The concentrations of the nutrients in the final mixture were: 0.6g/L NH4 +-N, 0.03g/L Fe2+0.2g/L of Mg2 +0.3g/L of K+0.05g/L of Ca2+。
Example 4
Preparing a culture solution with the ammonia nitrogen concentration of 300mg/L in the inlet water, and culturing nitrobacteria in a 100L biological aeration reaction tank by adopting an intermittent activated sludge method, wherein the temperature is 27 ℃, the pH value is 7.8, and the dissolved oxygen DO is 2.5-3.5 mg/L. Adding a growth promoter IV according to the concentration of the promoter in the culture system of 20 mg/L. And stopping culturing after the thalli enters a stable growth period, wherein the culturing time is 3 days, washing for many times after sedimentation or centrifugation, and collecting the thalli. Then, the cells were mixed with the prepared nutrient solution (the concentration of the nutrient was the same as in example 1) at a water content of 50%, shaken, and then 1.0% of dimethyl sulfoxide was added thereto, followed by storage in an ultra-low temperature freezer at-50 ℃. After 3 years, the activity and the survival rate of the thalli are taken out and examined, and after 2d recovery, the activity of the thalli can be completely recovered, the survival rate can reach 95 percent, and the ammonia nitrogen removal rate can reach more than 98 percent.
Example 5
Preparing a culture solution with the ammonia nitrogen concentration of 300mg/L in the inlet water, and culturing nitrobacteria in a 100L biological aeration reaction tank by adopting an intermittent activated sludge method, wherein the temperature is 27 ℃, the pH value is 7.8, and the dissolved oxygen DO is 2.5-3.5 mg/L. Adding growth promoter V according to the concentration of 20mg/L promoter in the culture system. And stopping culturing after the thalli enters a stable growth period, wherein the culturing time is 3 days, washing for many times after sedimentation or centrifugation, and collecting the thalli. Then, the cells were mixed with the prepared nutrient solution (the concentration of the nutrient was the same as in example 1) at a water content of 60%, shaken, and then 1.0% of dimethyl sulfoxide was added to the mixture, followed by storage in an ultra-low temperature freezer at-60 ℃. After 3 years, the activity and the survival rate of the thalli are taken out and examined, and after 2d recovery, the activity of the thalli can be completely recovered, the survival rate can reach 95 percent, and the ammonia nitrogen removal rate can reach more than 98 percent.
Comparative example 1
The processing mode and the operating conditions were the same as those of example 1 except that: no growth promoter is added during the culture of the nitrifying bacteria. The culture took 12 days to enter the stationary growth phase. After 3 years, the activity and the survival rate of the thalli are taken out and examined, and after 5d of recovery, the activity of the thalli can be completely recovered, the survival rate can reach 90 percent, and the ammonia nitrogen removal rate can reach 95 percent.
Comparative example 2
The processing mode and the operating conditions were the same as those of example 2 except that: no growth promoter is added during the culture of the nitrifying bacteria. It took 15 days to culture until entering stationary growth phase. After 5 years, the activity and the survival rate of the thalli are taken out and examined, and after 10 days of recovery, the activity of the thalli can be completely recovered, the survival rate can reach 85 percent, and the ammonia nitrogen removal rate can reach 90 percent.
Claims (9)
1. A preservation method of nitrifying bacteria is characterized by comprising the following steps:
(1) adding a growth promoter in the process of culturing nitrobacteria, wherein the growth promoter comprises 40-100 parts by weight of metal salt and 5-30 parts by weight of polyamine substance; the metal salt consists of calcium salt, copper salt, magnesium salt and/or ferrous salt; the polyamine substance is spermine, spermidine or a mixture of spermine and spermidine; after the culture reaches the growth stabilization period, collecting thalli;
(2) preparing a nitrifying bacteria preservation nutrient solution;
(3) mixing the nitrifying bacteria collected in the step (1) with the preservation nutrient solution prepared in the step (2), and controlling the water content to be 40-80%; NH in the mixture of the nitrifying bacteria and the preserved nutrient solution4 +-N concentration of 0.1-1.5g/L, Fe2+The concentration is 0.01-0.03g/L, K+Ca with a concentration of 0.05-0.5g/L2+The concentration is 0.01-0.05g/L, Mg2+The concentration is 0.05-0.25 g/L;
(4) adding a preservative;
(5) low-temperature freezing preservation at-20 deg.c to-70 deg.c.
2. The method of claim 1, wherein: culturing the nitrifying bacteria in the step (1) by adopting an intermittent activated sludge method; culturing nitrifying bacteria by adopting artificially prepared culture solution or ammonia nitrogen-containing wastewater; the culture solution mainly contains ammonium salt, and the ammonia nitrogen concentration is 200-1500 mg/L.
3. The method according to claim 1 or 2, characterized in that: culturing the nitrifying bacteria in the step (1) at the pH value of 7.0-8.5, the temperature of 20-40 ℃, the dissolved oxygen concentration of 1-5mg/L and the culturing time of 1-10 days; after the culture is finished, the obtained nitrifying bacteria are collected by a sedimentation, filtration or centrifugation method.
4. The method of claim 1, wherein: the metal salt in the growth promoter in the step (1) is calcium salt, magnesium salt and copper salt, wherein Ca2+、Mg2+And Cu2+The molar ratio of (5-15) to (5-25) to (0.5-5); or salts of calcium, ferrous and copper, where Ca2+、Fe2+And Cu2+The molar ratio of (5-15): (1-8): (0.5-5)(ii) a Or calcium, magnesium, ferrous and copper salts, of which Ca is present2+、Mg2+、Fe2+And Cu2+The molar ratio of (5-15): (5-25): 1-8): 0.5-5.
5. The method according to claim 1 or 4, characterized in that: the calcium salt in the growth promoter in the step (1) is CaSO4Or CaCl2Magnesium salt is MgSO4Or MgCl2The ferrous salt is FeSO4Or FeCl2The copper salt is CuSO4Or CuCl2。
6. The method of claim 1, wherein: adding 0.5-15 parts by weight of inorganic hydroxylamine acid into the growth promoter in the step (1).
7. The method of claim 6, wherein: the inorganic hydroxylamine acid is one or more of hydroxylamine hydrochloride, hydroxylamine sulfate or hydroxylamine phosphate, and the addition amount is 2-10 parts by weight.
8. The method of claim 1, wherein: the addition amount of the growth promoter in the step (1) is supplemented according to the concentration of the promoter in the culture system of 10-50 mg/L.
9. The method of claim 1, wherein: the preserving agent in the step (4) is dimethyl sulfoxide, and the using amount of the preserving agent is 0.5-3% of the volume of the mixture of the nitrifying bacteria and the preserving nutrient solution.
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Citations (3)
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| CN101899401A (en) * | 2009-05-25 | 2010-12-01 | 中国石油化工股份有限公司 | Microbial agent for treating ammonia-containing waste water and preparation method thereof |
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| CN103103143A (en) * | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Nitrifying bacteria preservation method |
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Patent Citations (3)
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| CN101899401A (en) * | 2009-05-25 | 2010-12-01 | 中国石油化工股份有限公司 | Microbial agent for treating ammonia-containing waste water and preparation method thereof |
| CN102381803A (en) * | 2010-09-06 | 2012-03-21 | 中国科学院城市环境研究所 | Method for starting full-flow autotrophic nitrogen removal process by taking general activated sludge as seed sludge |
| CN103103143A (en) * | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Nitrifying bacteria preservation method |
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