CN103773685A - Method for preserving nitrosomas - Google Patents
Method for preserving nitrosomas Download PDFInfo
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- CN103773685A CN103773685A CN201210404073.8A CN201210404073A CN103773685A CN 103773685 A CN103773685 A CN 103773685A CN 201210404073 A CN201210404073 A CN 201210404073A CN 103773685 A CN103773685 A CN 103773685A
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- nitrosomas
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- thalline
- nutritive medium
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Abstract
The invention discloses a method for preserving nitrosomas. The method comprises the following steps: (1) culturing nitrosomas to reach a nitrification rate of above 80%, and collecting the bacteria; (2) preparing a nitrosomas preservation nutrient solution; (3) mixing the nitrosomas bacteria collected in the step (1) with the nitrosomas preservation nutrient solution nitrite prepared in the step (2), adjusting the pH value to 7.5-8.5 and the moisture content to 40%-80%, wherein the moisture content rate refers to the weight content of water in the nitrosomas preservation system; (4) adding a preservative agent; and (5) conducting low temperature freezing preservation. Compared with the prior art, the invention has the advantages of simple method, high survival rate and fast activity recovery, and is suitable for long-term preservation of nitrosomas in a large amount.
Description
Technical field
The present invention relates to a kind of thalline method for preserving, be specifically related to a kind of method of simple and effective cryogenic freezing preservation Nitrosomas.
Background technology
Ammonia nitrogen is the pollutent that country realizes water pollution control overall control, in water body, the too high meeting of ammonia-nitrogen content causes Water Eutrophication, cause some algae excessive propagation in water body, other biological growth is affected, thereby destroy aquatic ecosystem, caused water quality deterioration and have influence on it using function.Bio-denitrification technology is the focus of the application of denitride technology research both at home and abroad at present with advantages such as its environment-friendly high-efficiency non-secondary pollutions.The nitrifying process of biological denitrificaion is by Nitromonas or Nitrosomas, ammonia nitrogen to be oxidized to nitre nitrogen or nitrite nitrogen under aerobic condition, then by the denitrifying bacteria under anoxia condition, nitre nitrogen or nitrite nitrogen is reduced into gaseous nitrogen and removes from water.In the middle of traditional biological denitrification process, wasted one nitrite nitrogen is transformed to nitre nitrogen, nitre nitrogen is converted into again the process of nitrite nitrogen.Short-cut nitrification and denitrification denitrogenation is that nitration reaction is controlled to the nitrite nitrogen stage, stops the further nitrated of nitrite nitrogen, then directly carries out denitrification.Compared with traditional nitration denitrification, short-cut nitrification and denitrification has advantages of as follows: (1) nitrated stage can reduce the oxygen requirement of 25% left and right, has reduced energy consumption; (2) the denitrification stage can reduce the organic carbon source of 40% left and right, has reduced working cost; (3) while reaction, ask shortening, reactor volume can reduce 30%～40% left and right; (4) there is higher denitrification rate; (5) sludge yield reduces; (6) reduced throwing alkali number etc.As can be seen here, control nitrifying process significant at Nitrification Stage, and after the High Performance Sub Nitromonas that completes short distance nitration selected, how permanently effective preservation is most important.
Microbial strains is one of important Biological resources, after a good bacterial classification is selected, must keep the constant or few slack-off change as much as possible of its good character, is just unlikely to reduce thalline performance, can prolonged application in production.The ultimate principle of culture presevation is mainly according to its physiological and biochemical property, manually creates conditions, and makes microbial metabolism in torpescence, the downtrod dormant state of growth and breeding, takes the conditions such as low temperature, dry, anoxic, makes bacterial classification temporarily in dormant state.First a kind of good method for preserving should be able to keep the original good character of bacterial classification constant for a long time, also needs the easy and economic of the method for considering itself simultaneously, to can promote the use of on producing.For conserving microorganism bacterial effectively, just need to select suitable method for preserving for the characteristic research of bacterial classification.Culture collection process is a lot, and at present both at home and abroad conventional have regular transfer methods, whiteruss method, sandy soil pipe method, vacuum freeze-drying method, Ultralow Temperature Freezer cold method, a ultra-low temperature liquid nitrogen cold method etc.Wherein regular transfer methods, whiteruss method, sandy soil pipe method, vacuum freeze-drying method are not suitable for the preservation of a large amount of bacterial classifications; Ultra-low temperature liquid nitrogen cold method and Ultralow Temperature Freezer cold method etc. all need to prepare cell suspension with protective material, and protective material has cow's milk, serum, carbohydrate, glycerine, methyl-sulphoxide etc., and object is to prevent because of the freezing or constantly infringement of distillation to cell of moisture.Freezing preservation is to solve a kind of effective way that germplasm is degenerated and prevented the sudden change of nature accumulation property, but traditional cryopreservation needs expensive programmed cooling instrument, complex steps.Vitrifying is a kind of novel method of cryopreservation completely; under high density protective material; cell all enters vitrifying state in fast cooling together with protective material; avoid intracellular ice crystal to form; make Organ and tissue each several part all enter identical state, have that equipment is simple, program is simple and the frozen advantage such as effective.
CN200810124325.5 provides a kind of culture collection process, adopts method semi-solid, that low temperature seal is cultivated: bacterial classification is inoculated in aseptic semisolid medium, pours in the envrionment temperature of vertically depositing 6～8 ℃ after sterile liquid paraffin and preserve.Described bacterial classification is streptococcus aureus, escherichia coli, Pseudomonas aeruginosa, subtilis, clostridium sporogenes, aspergillus niger or white chain pearl bacterium.Adopt this method can extend the shelf time of bacterial classification, the concentration of bacterial classification keeps relative stability simultaneously, has saved a large amount of costs for producing, testing, and has reduced processing and the discharge of waste after bacterial classification uses, and has reduced the harm to environment.But the method complex procedures is loaded down with trivial details, be not suitable for the long-term preservation of a large amount of thalline.
CN201010299763.2 provides a kind of freezing preservation erythrocytic method, to adding reductive agent and glycerine and ethylenediamine tetraacetic acid (EDTA) or edetate as metal ion chelation agent in red corpuscle; Reductive agent is any one or two or more the mixture in xitix or ascorbate salt, cysteine hydrochloride or N-acetylcystein, sodium borohydride, S-WAT or sodium bisulfite or Sodium Pyrosulfite, reduced glutathion, and reductive agent final concentration is respectively 0.5～300mM; The amount of glycerine counts 5%～10% with final volume ratio; The final concentration of ethylenediamine tetraacetic acid (EDTA) or edetate is 0.1～10mM; Fully mix and regulate acidity value to 6.5～8.0, by container closure, leave standstill freezing preservation below-40 ℃ after 1 hour.With the red corpuscle of the freezing preservation of the method, after 1 year, to thaw, erythroclasis rate is greater than 95%, and oxyphorase is without sex change, and not oxidized is methemoglobin.This kind of method is applicable to cell preserving process, is not suitable for the preservation of bacterial activity.
CN200810190852.6 relates to a kind of culture collection process, comprises the following steps: (1) is inoculated into the bacterial classification that needs preservation in sterilized bran mass; Culture medium prescription is husk, 1～2% SODIUMNITRATE, 1～2% urea, 1～2% ground rice, 0.2～1% vitamin H nutritional additive after 50～60% wheat brans, 35～45% are pulverized, and adds the deionized water of said mixture identical weight, mixes; (2) in the time that mycelia grows to the cell age needing, do not need to isolate mycelia and make bacteria suspension, directly freeze-drying liquid is added immediately in ampoul tube and mixed with culture; Freeze-drying liquid formula is 10～20 grams of skimmed milks, 0.2～0.5 gram of agar powder, 0.2～0.5 gram of reduced glutathion, 0.1～0.5 gram of gum arabic, 0.5～1 gram of trimethyl carbinol, with deionized water constant volume to 100 milliliter; (3) then lower the temperature with the rate of temperature fall of 2 ℃/min, lyophilize; (4) after freeze-drying, the ampoul tube of containing bacterium is vacuumized, then sealing; And preservation under low temperature inserted by obtained pipe by (5).This method adopts vacuum freeze-drying method preservation thalline, need to adopt slowly cooling process by thalline freeze-drying and then cryopreservation, complex steps, and energy consumption is higher.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of method for preserving of Nitrosomas.The present invention has the advantages such as method is simple, survival rate is high, activation recovering is quick, is suitable for a large amount of long-term preservation of Nitrosomas.
The method for preserving of Nitrosomas of the present invention, comprises the steps:
(1) Nitrosomas is cultured to nitrous rate and reaches more than 80%, and collect thalline;
(2) preparation Nitrosomas preservation nutritive medium;
(3) the nitrous acid thalline that step (1) is collected mixes with the Nitrosomas preservation nutritive medium of step (2) preparation, and pH is 7.5～9.0, and water ratio is 40%～80%, and water ratio refers to the weight content of water in Nitrosomas preservation system;
(4) add preserving agent;
(5) cryogenic freezing preservation.
In step of the present invention (1), Nitrosomas is cultivated the method that adopts this area routine, as adopted intermittent activated sludge process etc.Nitrosomas is cultivated the nutrient solution that can adopt human configuration, also can adopt waste water to cultivate Nitrosomas.Nutrient solution ingredient is mainly ammonium salt, and ammonia nitrogen concentration is 200～1500mg/L, also has a small amount of Fe
2+, Mg
2+, K
+, Ca
2+deng metal ion and phosphate anion etc.Nitrosomas culture condition is 30～40 ℃ of temperature, and pH is 7.0～8.5, and dissolved oxygen is 1.0～5.0mg/L, is cultured to that ammonia nitrogen removal frank reaches 99%, nitrous rate reaches 80% and collect thalline (nitrous rate refers to that mineralized nitrogen is the ratio of nitrite nitrogen) when above.Nitrosomas is cultured to nitrous rate and reaches 80% when above, collects nitrous acid thalline by sedimentation, filtration, the method such as centrifugal.The initial source that Nitrosomas is cultivated can be the Nitrosomas that needs arbitrarily preservation.
In step of the present invention (2), in Nitrosomas preservation nutritive medium, contain (NH
4)
2sO
4, contain micro substance FeSO simultaneously
4, KH
2pO
4, MgCl
2and CaCl
2.The consumption of various materials is determined by desired concn in step (3).
In step of the present invention (3), after Nitrosomas mixes with preservation nutritive medium, can adopt sodium bicarbonate, sodium carbonate and sodium hydroxide etc. to regulate pH.In Nitrosomas and preservation nutrient mix, NH
4 +-N concentration is 0.1～1.5g/L, Fe
2+concentration is 0.01～0.06g/L, K
+concentration is 0.05～0.5g/L, Ca
2+concentration is 0.01～0.1g/L, Mg
2+concentration is 0.05～0.5g/L.
In step of the present invention (4), described preserving agent can be the preserving agent of all routines of this area application, is preferably methyl-sulphoxide, consumption be Nitrosomas add volume of mixture after nutritive medium 2%～5%.
In step of the present invention (5), cryogenic freezing preservation can adopt the superfreeze equipment of this area routine.Cryogenic freezing preservation temperature is-20 ℃～-70 ℃.
The inventive method, by being optimized investigation from aspects such as yeast culture, water ratio, nutritive medium, protective materials, has obtained optimum Nitrosomas cryogenic freezing method for preserving.The inventive method can extend the shelf time of bacterial classification, makes the activity keeping of bacterial classification relatively stable simultaneously, and after thalline preservation, survival rate is high, and activation recovering is rapid, convenient for a large amount of thalline preservation economy, cost-saving for producing, testing.The inventive method is simple, does not need specific installation, convenient and swift, is applicable to a large amount of culture presevation, and survival rate can reach more than 90%.
Embodiment
A kind of specific implementation process of the present invention is as follows:
(1) utilize suitable nutrient solution, 30～40 ℃ of temperature, pH is 7.0～8.5, dissolved oxygen is 1.0～5.0mg/L, is cultured to that ammonia nitrogen removal frank reaches 99%, nitrous rate reaches 80% and collect thalline when above.
(2) water ratio of bacteria suspension is for the impact that is formed with of ice crystal inside and outside born of the same parents, and then affects preservation effect, therefore needs suitable water ratio.The initial water content of bacterial culture fluid is higher, therefore needs to reduce thalline water ratio.
(3) pH all has certain influence to controlling nitration reaction in the activity of Nitrification Stage and Nitrosomas, under cold condition, although thalline is in dormant state, but because nutritive substance is abundant, can make pH decline to some extent, therefore control the suitable pH of mixed solution most important for the preservation of thalline.
(4) nutritive substance is the necessary requirement of thalline existence, can make thalline preservation several years in cryogenic refrigerator, and not affect its activity, need to add preservation desired nutritional liquid according to suitable nutritive substance concentration.
(5) preserving agent can be all preserving agents of this area application, preferably methyl-sulphoxide C
2h
6sO does freezing agent.Methyl-sulphoxide is a kind of perviousness protective material, and to cell nontoxicity, molecular weight is little; solubleness is good; can penetrate rapidly cell, reduce freezing point, improve the permeability of cytolemma to water; delay freezing process; can make ICW before freezing, appear extracellular, form ice crystal outward born of the same parents, improve intracellular electrolyte concentration; reduce intracellular ice crystal, thereby reduce ice crystal to cell frostbite.Add the cryoprotectant of certain proportioning, usage quantity is to treat 2%～5% of preservation thalline volume after adding nutritive medium.
(6) cryogenic refrigerator is frozen: cryogenic freezing is between-20 ℃～-70 ℃.After 3～5 years abilities of preservation, investigate thalline survival rate and activity.
Embodiment 1
The nutrient solution that preparation influent ammonium concentration is 400mg/L adopts intermittent activated sludge process to cultivate Nitrosomas in 100L bio-aeration reaction tank, 32 ℃ of temperature, and pH is 7.8, dissolved oxygen is 1.0～2.5mg/L.When the ammonia nitrogen removal frank of thalline reaches more than 99%, nitrous rate reaches 80% and stops when above cultivating, sedimentation or centrifugal after repeatedly wash, reduce nitration product concentration.According to 50% water ratio, thalline is mixed with the nutritive medium of preparation, after shaking up, add 3% preserving agent methyl-sulphoxide, in-50 ℃ of cryogenic refrigerator preservations.Within 2 years, take out afterwards activity and the survival rate of investigating thalline, through the recovery of 3d, the activity of thalline can be recovered completely, and survival rate can reach 94%.
In Nitrosomas and preservation nutrient mix, the composition of nutritive substance and concentration are: ammonia nitrogen concentration is 0.4g/L, Fe
2+concentration is 0.02g/L, K
+concentration is 0.1g/L, Ca
2+concentration is 0.05g/L, Mg
2+concentration is 0.1g/L.
Embodiment 2
The nutrient solution that preparation influent ammonium concentration is 600mg/L adopts intermittent activated sludge process to cultivate Nitrosomas in 100L bio-aeration reaction tank, 35 ℃ of temperature, and pH is 8.0, dissolved oxygen is 1.5～3.0mg/L.When the ammonia nitrogen removal frank of thalline reaches more than 99%, nitrous rate reaches 80% and stops when above cultivating, sedimentation or centrifugal after repeatedly wash, reduce nitration product concentration.According to 60% water ratio, thalline is mixed with the nutritive medium of preparation, after shaking up, add 4% preserving agent methyl-sulphoxide, in-60 ℃ of cryogenic refrigerator preservations.Within 4 years, take out afterwards activity and the survival rate of investigating thalline, through the recovery of 5d, the activity of thalline can be recovered completely, and survival rate can reach 91%.
In Nitrosomas and preservation nutrient mix, the composition of nutritive substance and concentration are: ammonia nitrogen concentration is 0.6g/L, Fe
2+concentration is 0.02g/L, K
+concentration is 0.1g/L, Ca
2+concentration is 0.05g/L, Mg
2+concentration is 0.1g/L.
Embodiment 3
The nutrient solution that preparation influent ammonium concentration is 800mg/L adopts intermittent activated sludge process to cultivate Nitrosomas in 100L bio-aeration reaction tank, 37 ℃ of temperature, and pH is 8.5, dissolved oxygen is 1.5～2.5mg/L.When the ammonia nitrogen removal frank of thalline reaches more than 99%, nitrous rate reaches 80% and stops when above cultivating, sedimentation or centrifugal after repeatedly wash, reduce nitration product concentration.According to 70% water ratio, thalline is mixed with the nutritive medium of preparation, after shaking up, add 5% preserving agent methyl-sulphoxide, in-70 ℃ of cryogenic refrigerator preservations.Within 5 years, take out afterwards activity and the survival rate of investigating thalline, through the recovery of 7d, the activity of thalline can be recovered completely, and survival rate can reach 90%.
In Nitrosomas and preservation nutrient mix, the composition of nutritive substance and concentration are: ammonia nitrogen concentration is 0.6g/L, Fe
2+concentration is 0.02g/L, K
+concentration is 0.1g/L, Ca
2+concentration is 0.05g/L, Mg
2+concentration is 0.1g/L.
Claims (10)
1. a method for preserving for Nitrosomas, is characterized in that comprising the steps:
(1) Nitrosomas is cultured to nitrous rate and reaches more than 80%, and collect thalline;
(2) preparation Nitrosomas preservation nutritive medium;
(3) the nitrous acid thalline that step (1) is collected mixes with the Nitrosomas preservation nutritive medium of step (2) preparation, and pH is 7.5～8.5, and water ratio is 40%～80%, and water ratio refers to the weight content of water in Nitrosomas preservation system;
(4) add preserving agent;
(5) cryogenic freezing preservation.
2. method according to claim 1, is characterized in that: in step (1), Nitrosomas is cultivated and adopted intermittent activated sludge process.
3. method according to claim 1, it is characterized in that: in step (1), Nitrosomas culture condition is 30～40 ℃ of temperature, and pH is 7.0～8.5, dissolved oxygen is 1.0～5.0mg/L, is cultured to that ammonia nitrogen removal frank reaches 99%, nitrous rate reaches 80% and collect thalline when above.
4. according to the method described in claim 1,2 or 3, it is characterized in that: in step (1), Nitrosomas is cultured to nitrous rate and reaches 80% when above, collects nitrous acid thalline by sedimentation, filtration or centrifugal method.
5. method according to claim 1, is characterized in that: in step (2), in Nitrosomas preservation nutritive medium, contain (NH
4)
2sO
4, contain micro substance FeSO simultaneously
4, KH
2pO
4, MgCl
2and CaCl
2.
6. method according to claim 1, is characterized in that: in step (3), after Nitrosomas mixes with preservation nutritive medium, adopt sodium bicarbonate, sodium carbonate or sodium hydroxide to regulate pH.
7. method according to claim 1, is characterized in that: in step (3), and in Nitrosomas and preservation nutrient mix, NH
4 +-N concentration is 0.1～1.5g/L, Fe
2+concentration is 0.01～0.06g/L, K
+concentration is 0.05～0.5g/L, Ca
2+concentration is 0.01～0.1g/L, Mg
2+concentration is 0.05～0.5g/L.
8. method according to claim 1, is characterized in that: in step (4), preserving agent is methyl-sulphoxide.
9. according to the method described in claim 1 or 8, it is characterized in that: in step (4), preserving agent consumption be Nitrosomas add volume of mixture after nutritive medium 2%～5%.
10. method according to claim 1, is characterized in that: in step (5), cryogenic freezing preservation temperature is-20 ℃～-70 ℃.
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|---|---|---|---|
| CN201210404073.8A CN103773685B (en) | 2012-10-23 | 2012-10-23 | A kind of method for preserving of Nitrosomas |
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|---|---|---|---|
| CN201210404073.8A CN103773685B (en) | 2012-10-23 | 2012-10-23 | A kind of method for preserving of Nitrosomas |
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| Publication Number | Publication Date |
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| CN103773685A true CN103773685A (en) | 2014-05-07 |
| CN103773685B CN103773685B (en) | 2016-08-03 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554922A (en) * | 2015-09-30 | 2017-04-05 | 中国石油化工股份有限公司 | A kind of method for preserving of nitrite bacteria |
| CN108117993A (en) * | 2016-11-29 | 2018-06-05 | 中国石油化工股份有限公司 | A kind of method for preserving of denitrifying bacterium |
| CN108486019A (en) * | 2018-05-09 | 2018-09-04 | 浙江省农业科学院 | The method for preserving of nitrococcus |
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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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2012
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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 |
Non-Patent Citations (2)
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|---|
| （丹）艾雯 著: "《生物甲烷（上册）》", 30 June 2012 * |
| ZDENEK HUBÁLEK: "Protectants used in the cryopreservation of microorganisms", 《CRYOBIOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554922A (en) * | 2015-09-30 | 2017-04-05 | 中国石油化工股份有限公司 | A kind of method for preserving of nitrite bacteria |
| CN106554922B (en) * | 2015-09-30 | 2019-07-12 | 中国石油化工股份有限公司 | A kind of method for preserving of nitrite bacteria |
| CN108117993A (en) * | 2016-11-29 | 2018-06-05 | 中国石油化工股份有限公司 | A kind of method for preserving of denitrifying bacterium |
| CN108117993B (en) * | 2016-11-29 | 2021-05-04 | 中国石油化工股份有限公司 | Preservation method of denitrifying bacteria |
| CN108486019A (en) * | 2018-05-09 | 2018-09-04 | 浙江省农业科学院 | The method for preserving of nitrococcus |
| CN108486019B (en) * | 2018-05-09 | 2020-05-12 | 浙江省农业科学院 | Method for preserving nitrosobacteria |
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| Publication number | Publication date |
|---|---|
| CN103773685B (en) | 2016-08-03 |
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