CN106554930B - Culture method of low-temperature-resistant nitrifying bacteria and application of low-temperature-resistant nitrifying bacteria in wastewater treatment - Google Patents
Culture method of low-temperature-resistant nitrifying bacteria and application of low-temperature-resistant nitrifying bacteria in wastewater treatment Download PDFInfo
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Abstract
The invention discloses a method for culturing low-temperature-resistant nitrifying bacteria, which is characterized in that industrial ammonia-containing wastewater is used as a culture solution, activated sludge rich in nitrifying bacteria is subjected to enrichment culture in a manner of adding a nitrifying bacteria growth promoter, the culture process is performed in a manner of alternately performing normal-temperature culture at the temperature of 18-40 ℃ and low-temperature culture at the temperature of 8-18 ℃; the nitrobacteria growth promoter comprises 40-100 parts by weight of metal salt and 5-30 parts by weight of polyamine substance; the metal salt is composed of calcium salt, copper salt, magnesium salt and/or ferrous salt. The method is realized by adding the nitrobacteria growth promoter and matching with a normal-temperature and low-temperature alternate operation mode, the obtained thalli have wide application range and good treatment effect, can realize high-efficiency deamination and denitrification under the low-temperature condition, and can solve the problems of unstable low-temperature operation in winter, substandard sewage treatment and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method of low-temperature-resistant nitrifying bacteria and application of the low-temperature-resistant nitrifying bacteria in wastewater treatment.
Background
In the biological denitrification method, ammonia nitrogen is removed by nitrification of nitrifying bacteria in both traditional nitrification-denitrification, novel shortcut nitrification-denitrification and novel shortcut nitrification-anaerobic ammonia oxidation. Nitrifying bacteria belong to chemotrophic microorganisms, and biological cells can only utilize energy stored in forms of ATP and the like, but cannot directly utilize free energy released by chemical reactions. In aerobic metabolism, ATP is synthesized mainly by oxidative phosphorylation of the respiratory chain. The ammoxidation phosphorylation efficiency is very low, the ATP generated by the ammoxidation phosphorylation is very limited, and the energy is mainly used for electron transition to a higher energy level, so that the nitrobacteria grow very slowly, and the generation period is 8-36 h. The content of peptidoglycan in the cell wall of nitrobacteria is low, and the content of protein and fat is high, so that the nitrobacteria is sensitive to environmental change, the adaptability and tolerance of natural nitrobacteria in nature are poor, and the nitrobacteria cannot compete with heterotrophic microorganisms in growth under a plurality of conditions to obtain advantages. In a sewage treatment system, when the content of nitrifying bacteria in activated sludge is low, the nitrifying bacteria cannot grow and propagate quickly in a short time by adjusting the environmental conditions such as dissolved oxygen, pH and the like, and finally the ammonia nitrogen removal capability of the existing running sewage treatment system is limited. Particularly in winter in northern areas of China, the growth and reproduction rate and the biological activity of nitrobacteria in the activated sludge are greatly reduced due to the influence of low temperature, so that the nitrification and denitrification effects of a plurality of sewage treatment plants are poor, and the concentration of the effluent ammonia nitrogen is difficult to meet the discharge standard. The industrial application can generally adopt the method of directly feeding cultured low-temperature resistant nitrifying bacteria into a sewage treatment system to solve the problem. No matter nitrifying bacteria are cultured in the sewage treatment system directly or outside the sewage treatment system, substances for promoting growth need to be added to accelerate the growth rate of the nitrifying bacteria.
Chinese patents CN200510111874.5, CN200510111876.4, CN200510111877.9 and cn200510111875.x respectively propose nitrobacteria growth promoters composed of different metal salts, the main components of which include molasses, metal salts (iron salt, manganese salt, calcium salt and magnesium salt) and adsorbent. The ammonia nitrogen removal rate can be improved by more than 20 percent after the accelerator is used. However, the addition of the adsorbents mainly comprises substances such as zeolite powder, diatomite, powdered activated carbon or fly ash and the like, which inevitably increases the sludge yield.
Chinese patent CN201310296780.4 discloses enrichment of a low-temperature resistant autotrophic nitrifying bacteria agent and application thereof in wastewater treatment, which is characterized in that: the method comprises the steps of selecting seed sludge in a low-temperature reactor of a laboratory for domestication and enrichment, and specifically comprises the steps of carrying out enrichment culture on synthetic sewage without organic carbon and high ammonia nitrogen in three stages with different aeration quantities, and obtaining the autotrophic nitrifying bacteria with low-temperature resistance at the temperature of 10 ℃. Although the method solves the problem that part of sewage treatment plants which do not contain organic carbon run in winter, the enrichment culture solution used by the method does not contain organic carbon, so that the obtained nitrifying bacteria are limited when treating the waste water containing organic carbon.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a culture method of low temperature resistant nitrifying bacteria and application thereof in wastewater treatment. The method is realized by adding the nitrobacteria growth promoter and matching with a normal-temperature and low-temperature alternate operation mode, the obtained thalli have wide application range and good treatment effect, and the high-efficiency deamination and denitrification can be realized under the low-temperature condition. After the thalli which is subjected to enrichment culture is put into a sewage treatment plant for use, the problems of unstable low-temperature operation in winter, substandard sewage treatment and the like can be solved.
The method for culturing the low-temperature-resistant nitrifying bacteria takes industrial ammonia-containing wastewater as a culture solution, and adopts a method of adding a nitrifying bacteria growth promoter to perform enrichment culture on the activated sludge rich in the nitrifying bacteria, the domestication of the low-temperature-resistant nitrifying bacteria is performed in a mode of alternately performing normal-temperature culture and low-temperature culture in the culture process, the normal-temperature culture temperature is 18-40 ℃, the preferable temperature is 25-35 ℃, the low-temperature culture temperature is 8-18 ℃, and the preferable temperature is 10-15 ℃; the nitrobacteria growth promoter comprises 40-100 parts by weight of metal salt and preferably 50-80 parts by weight of polyamine substance, and 5-30 parts by weight of polyamine substance and preferably 10-20 parts by weight of polyamine substance; the metal salt is composed of calcium salt, copper salt, magnesium salt and/or ferrous salt.
The metal salt in the nitrobacteria growth promoter can be calcium salt, magnesium salt and copper salt, wherein Ca2+、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 nitrobacteria growth promoter is CaSO4Or CaCl2Preferably CaSO4(ii) a The magnesium salt is MgSO4Or Mg Cl2Preferably 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 nitrobacteria growth promoter is spermine, spermidine or a mixture of spermine and spermidine.
The nitrobacteria growth promoter can also comprise 0.5-15 parts by weight of inorganic hydroxylamine acid, preferably 2-10 parts by weight. The inorganic acid hydroxylamine is one or more of hydroxylamine hydrochloride, hydroxylamine sulfate or hydroxylamine phosphate, and is preferably hydroxylamine sulfate. 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 industrial ammonia-containing wastewater comprises all wastewater containing ammonia nitrogen and COD, wherein the ammonia nitrogen concentration is 100-500 mg/L, the COD (Cr method, the same below) concentration is 50-1000 mg/L, and the pH value is 6-9. The activated sludge rich in nitrobacteria can be activated sludge in all aeration tanks commonly used in the field, and preferably activated sludge treated with ammonia-containing sewage is inoculated according to the sludge sedimentation ratio of 20-40% in 30 minutes.
According to the enrichment culture method of nitrobacteria, the culture solution is replaced every batch, meanwhile, the nitrobacteria growth promoter is supplemented, and the nitrobacteria growth promoter is added according to the concentration of the promoter in a culture system of 10-50 mg/L, preferably 20-40 mg/L.
The method of alternately performing normal-temperature culture and low-temperature culture refers to performing culture for a certain time at normal temperature, preferably performing culture for 1-10 batches, and accelerating rapid propagation of nitrifying bacteria; then culturing for a certain time at a low temperature, preferably culturing for 1-10 batches, and domesticating the low temperature resistance of the nitrifying bacteria; and performing enrichment culture to obtain the low-temperature resistant nitrifying bacteria through alternative culture at normal temperature and low temperature. In the invention, preferably, in the normal-temperature culture process, when the removal rate of ammonia nitrogen of 3-5 continuous batches is more than 90% in the same culture time after the culture solution is replaced, the culture is switched to low-temperature culture, when the concentration of thalli of 3-5 continuous batches is not reduced any more in the low-temperature culture process, the culture is switched to normal-temperature culture, the normal-temperature culture and the low-temperature culture are alternately carried out for 2-6 times, the culture in one period is finished, and thalli are harvested. The conditions of normal temperature culture and low temperature culture are as follows: the dissolved oxygen concentration is 1-5 mg/L, preferably 2-3 mg/L; the pH is 7.0 to 9.0, preferably 7.8 to 8.5.
The application of the low temperature resistant nitrifying bacteria subjected to enrichment culture in wastewater treatment can be used for treating various industrial ammonia-containing wastewater, wherein the ammonia nitrogen concentration is 100-1000 mg/L, and the COD concentration is 50-1000 mg/L; the wastewater treatment temperature is 5-35 ℃, preferably 10-30 ℃, the dissolved oxygen concentration is 1-5 mg/L, and the pH is 7.0-9.0. After treatment, the highest nitration ratio degradation rate can reach 10mgNH3N/(g MLSS h), exceeding the nitrification effect of low temperature resistant nitrifiers in the general literature. After the thalli which is subjected to enrichment culture is put into a sewage treatment plant for use, the problems of unstable low-temperature operation in winter, substandard sewage treatment and the like can be solved.
Compared with the prior art, the invention has the following advantages:
1. according to the invention, the nitrobacteria which are propagated in large quantities at normal temperature are subjected to low-temperature domestication continuously by adding the nitrobacteria growth promoter and matching with a normal-temperature and low-temperature alternate culture mode, the temperature change adapting capacity of the thalli is obviously enhanced, the obtained thalli has wide adapting range and good treatment effect, and high-efficiency ammonia nitrogen removal can be realized under a low-temperature condition.
2. The growth promoter used in the invention contains metal ions provided by metal salts as elements required by the growth of nitrobacteria, and simultaneously, the metal ions are used as enzyme components to improve the activity of the enzyme, thereby improving the rapid degradation of substrates by thalli and accelerating the process of enzymatic reaction. The polyamine can promote cell proliferation, and can accelerate cell proliferation and prolong the service life of thallus under the combined action of the polyamine and metal ions.
3. According to the invention, the nitrobacteria growth promoter with special composition and proportion is added in the nitrobacteria enrichment culture process, so that the nitrobacteria can quickly adapt to a low-temperature environment under the combined action of metal salt, polyamine substances and inorganic hydroxylamine sulfate, can grow and reproduce under a low-temperature condition, can realize good synergy with an activated sludge system in a sewage treatment system, and improve the ammonia nitrogen removal effect of the sewage plant in winter during low-temperature operation.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Firstly, preparing a metal salt solution according to the proportion and the formula of the nitrobacteria 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 nitrobacteria growth promoters I-IV, wherein the concentration of the promoters is 0.5 g/L.
TABLE 1 formulation and proportion of nitrobacteria growth promoters
Example 1
Activated sludge rich in nitrobacteria of a certain sewage treatment plant is inoculated into the nitrobacteria culture reactor according to the inoculation amount of 20 percent (30 minutes of sludge sedimentation ratio), and the water quality of the industrial ammonia-containing wastewater is as follows: the ammonia nitrogen concentration is 100-150 mg/L, the COD is 50-100 mg/L, and the pH is 7-8. And (3) replenishing the nitrobacteria growth promoter I while replacing the culture solution in each batch, and adding the nitrobacteria growth promoter I according to the concentration of the promoter in the culture system of 20 mg/L. The culture process adopts a mode of alternately carrying out normal temperature culture and low temperature culture, firstly, 1 batch of the low temperature culture is carried out at the temperature of 25 ℃, then, 1 batch of the low temperature culture is carried out at the temperature of 15 ℃, the normal temperature culture and the low temperature culture are changed into the normal temperature culture, the normal temperature culture and the low temperature culture are alternately carried out for 10 times, the culture process of one period is ended, and the low temperature resistant nitrobacteria A are harvested. The concentration of dissolved oxygen during the culture process was 2.0mg/L, and the pH was 7.8.
Example 2
Activated sludge rich in nitrobacteria of a certain sewage treatment plant is inoculated into the nitrobacteria culture reactor according to the inoculation amount of 25 percent (30-minute sedimentation ratio of the sludge), and the water quality of the industrial ammonia-containing wastewater is as follows: the ammonia nitrogen concentration is 200-250 mg/L, the COD is 200-500 mg/L, and the pH is 7.5-8.3. And (3) replacing the culture solution in each batch, simultaneously replenishing the nitrobacteria growth promoter II, and adding according to the concentration of the promoter in the culture system of 25 mg/L. The culture process adopts a mode of alternately carrying out normal-temperature culture and low-temperature culture, firstly, 2 batches of the low-temperature culture are carried out at the temperature of 30 ℃, then, 2 batches of the low-temperature culture are carried out at the temperature of 12 ℃, the normal-temperature culture and the low-temperature culture are changed into the normal-temperature culture, the normal-temperature culture and the low-temperature culture are alternately carried out for 6 times, the culture process of one period is ended, and the low-temperature resistant nitrifying bacteria B are harvested. The concentration of dissolved oxygen during the culture process was 2.5mg/L, and the pH was 8.0.
Example 3
Activated sludge rich in nitrobacteria of a certain sewage treatment plant is inoculated into a nitrobacteria culture reactor according to the inoculation amount of 30 percent (30 minutes of sludge sedimentation ratio), and the water quality of the industrial ammonia-containing wastewater is as follows: the ammonia nitrogen concentration is 300-400 mg/L, the COD is 500-600 mg/L, and the pH is 7.5-8.5. And (3) replenishing a nitrobacteria growth promoter III while replacing the culture solution in each batch, and adding the nitrobacteria growth promoter III according to the concentration of the promoter in the culture system of 30 mg/L. The culture process adopts a mode of alternately carrying out normal temperature culture and low temperature culture, firstly, 3 batches of the culture are carried out at normal temperature under the condition of the temperature of 35 ℃, then, 4 batches of the culture are carried out at low temperature under the condition of the temperature of 15 ℃, then, the culture is carried out at normal temperature, the normal temperature culture and the low temperature culture are alternately carried out for 4 times, the culture process of one period is ended, and the low temperature resistant nitrifying bacteria C are harvested. The concentration of dissolved oxygen during the culture process was 2.0mg/L, and the pH was 8.5.
Example 4
Activated sludge rich in nitrobacteria of a certain sewage treatment plant is inoculated into the nitrobacteria culture reactor according to the inoculation amount of 35 percent (30-minute sedimentation ratio of the sludge), and the water quality of the industrial ammonia-containing wastewater is as follows: the ammonia nitrogen concentration is 400-500 mg/L, the COD is 900-960 mg/L, and the pH is 7-9. And (3) replenishing the nitrobacteria growth promoter IV while replacing the culture solution in each batch, and adding the nitrobacteria growth promoter IV according to the concentration of the promoter in the culture system of 40 mg/L. The culture process adopts a mode of alternately carrying out normal temperature culture and low temperature culture, firstly carrying out normal temperature culture for 3 batches at the temperature of 25 ℃, then carrying out low temperature culture for 3 batches at the temperature of 15 ℃, then carrying out normal temperature culture, carrying out the normal temperature culture and the low temperature culture for 5 times alternately, finishing the culture process of one period, and harvesting the low temperature resistant nitrifying bacteria D. The concentration of dissolved oxygen during the culture process was 3.0mg/L, and the pH was 8.0.
Example 5
The processing technique and the operating conditions are the same as those in example 3, except that in the normal-temperature culture process, when the removal rate of ammonia nitrogen in 3 continuous batches is more than 90% after the culture solution is replaced, the culture is switched to low-temperature culture, when the concentration of the bacteria in 4 continuous batches is not reduced any more in the low-temperature culture process, the culture at normal temperature and low temperature is alternately carried out for 4 times, the culture in one period is finished, and the low-temperature resistant nitrifying bacteria E are obtained.
Example 6
The processing technique and the operating conditions are the same as those in example 4, except that in the normal-temperature culture process, when the removal rate of ammonia nitrogen in 3 continuous batches is more than 90% after the culture solution is replaced, the culture is switched to low-temperature culture, when the concentration of the bacteria in 3 continuous batches is not reduced any more in the low-temperature culture process, the culture is switched to normal-temperature culture, the normal-temperature culture and the low-temperature culture are alternately carried out for 5 times, the culture in one period is finished, and the low-temperature resistant nitrifying bacteria F are obtained.
Comparative example 1
The processing technique and the operation conditions are the same as those in example 4, except that the culture process does not adopt a mode of alternately carrying out normal temperature culture and low temperature culture, and the temperature is always 25 ℃. After the culture is finished, the nitrifying bacteria G are harvested.
Comparative example 2
The processing technique and the operation conditions are the same as those in example 4, except that the culture process does not adopt a mode of alternately carrying out normal temperature culture and low temperature culture, and the temperature is always 15 ℃. After the culture is finished, the nitrifying bacteria H are harvested.
Comparative example 3
The treatment process and the operating conditions were the same as in example 4, except that: no nitrobacteria growth promoter is added in the culture process. After the culture is finished, the nitrifying bacteria I are harvested.
The nitrifying bacteria A-I obtained by the culture are used for treating catalyst ammonia-containing wastewater, wherein the ammonia nitrogen concentration is 500mg/L, the COD concentration is 100mg/L, the wastewater treatment temperature is 10 ℃, the dissolved oxygen concentration is 2-3 mg/L, and the pH is 7.5-8.0. The ammonia nitrogen removal rate in the same reaction time is shown in Table 2.
TABLE 2 treatment effect of low temperature resistant nitrifying bacteria
As can be seen from Table 2, when the low temperature resistant nitrifying bacteria A-F are used for treating ammonia-containing wastewater at low temperature, the removal rates of ammonia and nitrogen are both more than 85%, the nitrifying bacteria I are obtained without adding a nitrifying bacteria growth promoter, and the removal rates of ammonia and nitrogen are both less than 50% for the nitrifying bacteria G and H which are obtained by only culturing at normal temperature and only culturing at low temperature. Therefore, the culture of the low-temperature resistant nitrobacteria can achieve good deamination and denitrification effects only by combining the use of the growth promoter and the normal-temperature and low-temperature alternate culture mode. Therefore, after the thalli subjected to enrichment culture is added into a sewage treatment plant for use, the problems of unstable low-temperature operation in winter, substandard sewage treatment and the like can be solved.
Claims (10)
1. A method for culturing low-temperature resistant nitrifying bacteria is characterized by comprising the following steps: taking industrial ammonia-containing wastewater as a culture solution, and performing enrichment culture on the activated sludge rich in nitrobacteria by adopting a method of adding a nitrobacteria growth promoter, wherein the culture process adopts a mode of alternately performing normal-temperature culture and low-temperature culture, the normal-temperature culture temperature is 18-40 ℃, and the low-temperature culture temperature is 8-18 ℃; the nitrobacteria growth promoter comprises 40-100 parts by weight of metal salt and 5-30 parts by weight of polyamine substance, wherein the metal salt consists of calcium salt, copper salt, magnesium salt and/or ferrous salt; the polyamine substances in the growth promoter are spermine, spermidine or a mixture of spermine and spermidine; the metal salt in the growth promoter is calcium salt, magnesium salt and copper salt, wherein Ca is2+、Mg2+And Cu2+In a molar ratio of (5-15): (5-25): 0.5-5), or calcium salt, ferrous salt and copper salt, wherein Ca is2+、Fe2+And Cu2+In a molar ratio of (5-15): (1-8): 0.5-5), or calcium salt, magnesium salt, ferrous salt and copper salt, wherein Ca is2+、Mg2+、Fe2+And Cu2+The molar ratio of (5-15): (5-25): (1-8): 0.5-5).
2. The method of claim 1, wherein: the calcium salt in the growth promoter is CaSO4Or CaCl2Magnesium salt is MgSO4Or MgCl2The ferrous salt is FeSO4Or FeCl2The copper salt is CuSO4Or CuCl2。
3. The method of claim 1, wherein: the growth promoter also comprises 0.5-15 parts by weight of inorganic hydroxylamine acid.
4. The method of claim 3, wherein: the inorganic hydroxylamine acid is one or more of hydroxylamine hydrochloride, hydroxylamine sulfate or hydroxylamine phosphate, and the content is 2-10 parts by weight.
5. The method of claim 1, wherein: the industrial ammonia-containing wastewater comprises all wastewater containing ammonia nitrogen and COD, wherein the ammonia nitrogen concentration is 100-500 mg/L, the COD concentration is 50-1000 mg/L, and the pH value is 6-9; the activated sludge rich in nitrobacteria is activated sludge treated with ammonia-containing sewage, and is inoculated according to the sludge sedimentation ratio of 20-40% in 30 minutes.
6. The method of claim 1, wherein: in the enrichment culture method of nitrobacteria, the culture solution is replaced every batch, meanwhile, the nitrobacteria growth promoter is supplemented, and the nitrobacteria growth promoter is added according to the concentration of the promoter in a culture system of 10-50 mg/L.
7. The method of claim 1, wherein: the mode of alternately carrying out normal-temperature culture and low-temperature culture refers to carrying out 1-10 batches of culture at normal temperature to accelerate the rapid propagation of nitrobacteria; then culturing 1-10 batches at low temperature, and domesticating the low temperature resistance of the nitrifying bacteria; and performing enrichment culture to obtain the low-temperature resistant nitrifying bacteria through alternative culture at normal temperature and low temperature.
8. The method of claim 7, wherein: and in the normal-temperature culture process, when the removal rate of ammonia nitrogen of 3-5 continuous batches is more than 90% in the same culture time after the culture solution is replaced, the culture is switched to low-temperature culture, when the concentration of the bacteria of 3-5 continuous batches is not reduced any more in the low-temperature culture process, the culture is switched to normal-temperature culture, the normal-temperature culture and the low-temperature culture are alternately carried out for 2-6 times, the culture in one period is finished, and the bacteria are harvested.
9. The method of claim 1, wherein: the normal temperature culture temperature is 25-35 ℃, and the low temperature culture temperature is 10-15 ℃; the concentration of dissolved oxygen in the normal temperature culture and the low temperature culture is 1-5 mg/L, and the pH is 7.0-9.0.
10. The use of low temperature resistant nitrifying bacteria enriched and cultured by the method of any one of claims 1 to 9 in wastewater treatment, wherein: the low temperature resistant nitrifying bacteria are used for treating various industrial ammonia-containing wastewater, wherein the ammonia nitrogen concentration is 100-1000 mg/L, the COD concentration is 50-1000 mg/L, the wastewater treatment temperature is 5-35 ℃, the dissolved oxygen concentration is 1-5 mg/L, and the pH is 7.0-9.0.
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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 |
| CN104164386A (en) * | 2014-07-02 | 2014-11-26 | 中国科学院成都生物研究所 | Method of medium-temperature enrichment culturing and low-temperature application of low-temperature resistant nitrobacterium flora |
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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 |
| CN104164386A (en) * | 2014-07-02 | 2014-11-26 | 中国科学院成都生物研究所 | Method of medium-temperature enrichment culturing and low-temperature application of low-temperature resistant nitrobacterium flora |
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