CN106554922B - A kind of method for preserving of nitrite bacteria - Google Patents
A kind of method for preserving of nitrite bacteria Download PDFInfo
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- CN106554922B CN106554922B CN201510635144.9A CN201510635144A CN106554922B CN 106554922 B CN106554922 B CN 106554922B CN 201510635144 A CN201510635144 A CN 201510635144A CN 106554922 B CN106554922 B CN 106554922B
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Abstract
The invention discloses a kind of method for preserving of nitrite bacteria, including (1) adds growth promoter in nitrite bacteria incubation, and the promotor includes metal salt, polyamines, organic acid azanol and Na2SO3, the metal salt includes calcium salt, mantoquita, magnesium salts and/or ferrous salt；Culture to ammonia nitrogen removal frank up to 99%, nitrosoation rate up to 80% when, collect thallus；(2) nitrite bacteria preservation nutrient solution is prepared；(3) nitrite bacteria that step (1) is collected is mixed with the preservation nutrient solution that step (2) is prepared, control pH is 7.5-9.0, moisture content 40%-80%；(4) preserving agent is added；(5) cryogenic freezing preservation.The present invention adds growth promoter in nitrite bacteria incubation, can make thallus fast breeding, improves nitrosoation rate, shortens incubation time；Microbial activity and low temperature tolerance ability especially can be improved, it being capable of quick activity recovery after low temperature cold cold storage.
Description
Technical field
The present invention relates to a kind of thallus method for preserving, and in particular to a kind of method of cryogenic freezing preservation nitrite bacteria.
Background technique
Strain is one of main living resources, after an excellent strain is selected, it is necessary to keep its excellent
Character is constant or lacks slack-off change as much as possible, is just unlikely to reduce thallus performance, can apply in production for a long time.Culture presevation side
Method has very much, and freezen protective is a kind of effective way for solving germplasm and degenerating and preventing nature accumulation property mutation, but traditional low
Temperature, which saves, needs expensive programmed cooling instrument device, complex steps.Complete vitrifying is a kind of new method of cryo-conservation, i.e., in height
Under concentration protective agent, cell all enters glassy state together with protective agent in fast cooling, avoids intracellular ice crystal from being formed, makes device
Official and tissue each section all enter identical state, than other Cryopreservations are simple with equipment, program is simple and freezes
The advantages that effect is good has distinctive feature in terms of saving the structural intergrity of organ and tissue.
Mainly according to microbial physiology Biochemical Characteristics, artificial creation's condition makes the basic principle of Microbiological Culture Collection
Microbial metabolism is in the suppressed dormant state of torpescence, growth and breeding, that is, takes the conditions such as low temperature, drying, anoxic, make bacterium
Kind is temporary in a dormant state.A kind of good method for preserving should be able to keep the original merit of strain constant for a long time first, together
When it is also necessary to take into account that method itself easily and economically, so as to produce it is upper can promote the use of.Therefore, suitable preservation side is being determined
Under the premise of method, the cold tolerance of thallus how is improved, for maintaining the excellent performance of thallus, preventing spawn degeneration that there is weight
Want meaning.
It by Nitromonas or nitrite bacteria by ammonium oxidation is nitre that the nitrifying process of biological denitrificaion, which is under aerobic condition,
Nitrogen or nitrite nitrogen, then nitrate nitrogen or nitrite nitrogen are reduced into from water by gaseous nitrogen by the denitrifying bacterium under anoxia condition and removed.It passes
One being wasted among system biological denitrification process, nitrite nitrogen being converted into nitrate nitrogen, nitrate nitrogen is converted into the process of nitrite nitrogen again.Short distance nitre
Changing denitrification denitrogenation is to control nitration reaction in the nitrite nitrogen stage, prevents the further nitrification of nitrite nitrogen, is then directly carried out
Denitrification.Compared with traditional nitration denitrification, short-cut nitrification and denitrification has the advantage that (1) nitrification stage can reduce 25%
The oxygen demand of left and right, reduces energy consumption；(2) the denitrification stage can reduce by 40% or so organic carbon source, reduce operating cost；
(3) shortening is asked when reacting, and reactor volume can reduce 30%-40% or so；(4) denitrification rate with higher；(5) sludge produces
Amount reduces；(6) reduce and throw alkali number etc..It can be seen that control nitrifying process is of great significance in Nitrification Stage, and complete
After the high-performance nitrite bacteria of short distance nitration is selected, how permanently effective preservation is most important.
CN201210404073.8 discloses a kind of method for preserving of nitrite bacteria, comprising: ((1) is by nitrite bacteria
Culture collects thallus to nitrosoation rate up to 80% or more；(2) nitrite bacteria preservation nutrient solution is prepared；(3) step (1) is collected
Nitrite bacteria body mixed with the nitrite bacteria preservation nutrient solution that step (2) is prepared, pH 7.5-9.0, moisture content is
40%-80%, moisture content refer to the weight content of water in nitrite bacteria preservation system；(4) preserving agent is added；(5) cryogenic freezing is protected
Hiding.This method can extend the holding time of strain, while the activity of strain being made to keep opposite stabilization.But due to nitrous acid
Bacterial growth is slower, needs Spawn incubation is longer to the period of growth stationary phase.In addition, thallus is in cryopreservation procedures,
Need to add high concentration nutritional substance and a large amount of preserving agents, preservation higher cost.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of method for preserving of nitrite bacteria.The present invention is in nitrous
Growth promoter is added in acetic bacterial incubation, thallus fast breeding can be made, improves nitrosoation rate, shortens incubation time；
Microbial activity and low temperature tolerance ability especially can be improved, after low temperature cold cold storage can quick activity recovery, preservation cost compared with
It is low.
The method for preserving of nitrite bacteria of the present invention, includes the following steps:
(1) growth promoter is added in nitrite bacteria incubation, the growth promoter includes metal salt, polyamines
Substance, organic acid azanol and Na2SO3, the metal salt includes calcium salt, mantoquita, magnesium salts and/or ferrous salt；Culture to ammonia nitrogen is gone
When except rate up to 99%, nitrosoation rate up to 80% or more, thallus is collected；
(2) nitrite bacteria preservation nutrient solution is prepared；
(3) nitrite bacteria that step (1) is collected is mixed with the preservation nutrient solution that step (2) is prepared, control pH is
7.5-9.0, moisture content 40%-80%, preferably 50%-70%；
(4) preserving agent is added；
(5) cryogenic freezing preservation.
The culture of nitrite bacteria described in step (1) of the present invention is adopted with the conventional methods in the field, such as using intermittent
Activated sludge process etc..The culture solution of human configuration can be used, nitrite bacteria can also be trained using nitrogen-containing wastewater
It supports, culture solution ingredient is mainly ammonium salt, ammonia nitrogen concentration 200-1500mg/L.The primary source of nitrite bacteria culture can
To be arbitrary the nitrite bacteria for needing preservation.The condition of culture of nitrite bacteria are as follows: pH value 7.0-8.5, preferably
8.0-8.5；Temperature is 30-40 DEG C, preferably 35-38 DEG C；Dissolved oxygen concentration is 1-5mg/L, preferably 2-3mg/L.When culture extremely
When ammonia nitrogen removal frank is up to 99%, nitrosoation rate (nitrosoation rate refers to that mineralized nitrogen is the ratio of nitrite nitrogen) up to 80%, by sedimentation,
Nitrous acid thallus is collected in the methods of filtering, centrifugation.
Metal salt in step (1) the of the present invention growth promoter is 40-100 parts by weight, preferably 50-80 weight
Part, polyamines are 5-30 parts by weight, preferably 10-20 parts by weight, and organic acid azanol is 0.05-1.5 parts by weight, preferably
0.1-1.0 parts by weight, Na2SO3For 10-40 parts by weight, preferably 20-30 parts by weight.
Metal salt in step (1) the of the present invention growth promoter includes calcium salt, mantoquita, magnesium salts and/or ferrous salt, such as
It can be calcium salt, ferrous salt and mantoquita, Ca2+、Fe2+And Cu2+Molar ratio be (5-15): (1-8): (0.5-5), preferably (8-
12): (2-6): (1-4)；Either calcium salt, magnesium salts and mantoquita, wherein Ca2+、Mg2+And Cu2+Molar ratio be (5-15): (5-
25): (0.5-5), preferably (8-12): (10-20): (1-4)；Either calcium salt, magnesium salts, ferrous salt and mantoquita, Ca2+、Mg2+、
Fe2+And Cu2+Molar ratio be (5-15): (5-25): (1-8): (0.5-5), preferably (8-12): (10-20): (2-6): (1-
4).
Calcium salt in step (1) the of the present invention growth promoter is CaSO4Or CaCl2, preferably CaCl2；Magnesium salts is
MgSO4Or MgCl2, preferably MgCl2；Mantoquita is CuSO4Or CuCl2, preferably CuCl2；Molysite is FeSO4Or FeCl2, excellent
Select FeCl2。
Polyamines described in step (1) of the present invention are the mixture of spermine, spermidine or both.Described is organic
Sour azanol is the mixture of formic acid azanol, hydroxylamine acetate or both.
The additional amount of growth promoter described in step (1) of the present invention is according to promoter concentration 10-50mg/ in cultivating system
L is added, and preferably 20-30mg/L is added.
The ammonium salt contained in nitrite bacteria preservation nutrient solution described in step (2) of the present invention is (NH4)2SO4, also contain
Micro substance FeSO4、KH2PO4、MgCl2And CaCl2.The dosage of various substances is determined by concentration required in step (3).
In step (3) of the present invention, after nitrite bacteria mixes with preservation nutrient solution, sodium bicarbonate, sodium carbonate can be used
Or sodium hydroxide etc. adjusts pH.PH all has a fixing in the activity of Nitrification Stage and nitrite bacteria to control nitration reaction
It rings, therefore it is most important for the long term storage of nitrite bacteria to control the suitable pH of mixed liquor.Nitrite bacteria and preservation are sought
In nutrient solution mixture, NH4 +- N concentration is 0.1-1.5g/L, Fe2+Concentration is 0.01-0.03g/L, K+Concentration is 0.05-0.5g/
L, Ca2+Concentration is 0.01-0.05g/L, Mg2+Concentration is 0.05-0.25g/L.The moisture content of bacteria suspension is for outer ice crystal intracellular
Formation has an impact, and then influences preservation effect, it is therefore desirable to which suitable moisture content, the moisture content that the present invention controls preservation system are
40%-80%, preferably 50%-70%, moisture content refer to the weight content of water in nitrite bacteria preservation system.
Preserving agent described in step (4) of the present invention can be all conventional preserving agents of this field application, preferably diformazan
Base sulfoxide, usage amount are the 0.5%-3% of nitrobacteria and preservation nutrient mix volume.Dimethyl sulfoxide is a kind of permeability guarantor
Agent is protected, to cytotoxic, molecular weight is small, and solubility is good, can penetrate cell rapidly, reduces freezing point, improves cell membrane to water
Permeability delays freezing process, and intracellular water can be made to appear before freezing extracellularly, in extracellular formation ice crystal, improved thin
Electrolyte concentration intracellular reduces intracellular ice crystal, to reduce ice crystal to cell frostbite.
In step (5) of the present invention, cryogenic freezing preservation temperature is between -20 DEG C~-70 DEG C.It can be conventional using this field
Freezing equipment.
Compared with prior art, the invention has the following advantages:
1, the present invention specific growth promoter is added in nitrite bacteria incubation so that thallus metal salt,
Polyamines, organic acid azanol and Na2SO3Collective effect under realize fast breeding, improve nitrosoation rate, shorten training
The time is supported, solves the problems, such as that nitrite bacteria increasess slowly.
2, the addition of growth promoter can provide good nutrient environment to somatic cells, can be improved institute's preservation thallus
Stability and freezing tolerance, extend the holding time of strain.Thallus after low temperature cold cold storage can be quickly extensive in a short time
Active, survival rate is high, tolerance impact capacity is strong.
3, growth promoter is added when nitrite bacteria is cultivated, metal salt therein also acts as while being both nutrient solution
The effect of protective agent buffer solution reduces the usage amount of preserving agent, reduces the activity suppression to strain.
Specific embodiment
The following describes the present invention in detail with reference to examples.But therefore do not cause limitation of the present invention.
Metal salt solution is prepared first, in accordance with the ratio and formula of the growth promoter of table 1, it is preceding by more amines using
Matter, organic acid acid azanol and Na2SO3It is added in metal salt solution, the nitrite bacteria growth that different model is prepared promotes
Agent I-III, the promoter concentration are 0.5g/L.
The formula and ratio of 1 nitrite bacteria growth promoter of table
Embodiment 1
The culture solution that influent ammonium concentration is 400mg/L is prepared, intermittent work is used in 100L bio-aeration reaction tank
Property sludge nitrite bacteria is cultivated, 32 DEG C of temperature, pH 7.8, dissolved oxygen DO be 1.0-2.5mg/L.According to culture
Growth promoter I is added in promoter concentration 25mg/L in system.When the ammonia nitrogen removal frank of thallus is up to 99% or more, nitrosoation rate reaches
Stop culture when 80%, incubation time is 3 days, is repeatedly washed after sedimentation or centrifugation, collects thallus.Then contain according to 50%
Water rate mixes thallus with the preservation nutrient solution of preparation, and 1.5% preserving agent dimethyl sulfoxide is added after shaking up, super in -50 DEG C
Low temperature refrigerator preservation.The activity and survival rate for investigating thallus are taken out after 3 years, by the recovery of 2d, the activity of thallus can be completely extensive
Multiple, survival rate is up to 95%, and nitrosoation rate is up to 80% or more.
Concentration of the nutriment in final mixture are as follows: the NH of 0.4g/L4 +The Fe of-N, 0.01g/L2+, 0.05g/L's
Mg2+, the K of 0.1g/L+, the Ca of 0.01g/L2+, adjusting pH is 8.5.
Embodiment 2
The culture solution that influent ammonium concentration is 600mg/L is prepared, intermittent work is used in 100L bio-aeration reaction tank
Property sludge nitrite bacteria is cultivated, 35 DEG C of temperature, pH 8.0, dissolved oxygen DO be 1.5-3.0mg/L.According to culture
Growth promoter II is added in promoter concentration 30mg/L in system.When the ammonia nitrogen removal frank of thallus is up to 99% or more, nitrosoation rate reaches
Stop culture when 80%, incubation time is 5 days, is repeatedly washed after sedimentation or centrifugation, collects thallus.Then contain according to 60%
Water rate mixes thallus with the nutrient solution of preparation, and 2.0% preserving agent dimethyl sulfoxide is added after shaking up, ultralow in -60 DEG C
Temperature refrigerator preservation.The activity and survival rate for investigating thallus are taken out after 4 years, by the recovery of 3d, the activity of thallus can be restored completely,
Survival rate is up to 90%, and nitrosoation rate is up to 80% or more.
Concentration of the nutriment in final mixture are as follows: the NH of 0.6g/L4 +The Fe of-N, 0.01g/L2+, 0.05g/L's
Mg2+, the K of 0.1g/L+, the Ca of 0.01g/L2+, adjusting pH is 8.0.
Embodiment 3
The culture solution that influent ammonium concentration is 800mg/L is prepared, intermittent work is used in 100L bio-aeration reaction tank
Property sludge nitrite bacteria is cultivated, 37 DEG C of temperature, pH 8.5, dissolved oxygen DO be 1.5-2.5mg/L.According to culture
Growth promoter III is added in promoter concentration 20mg/L in system.When the ammonia nitrogen removal frank of thallus is up to 99% or more, nitrosoation rate reaches
Stop culture when 80%, incubation time is 7 days, is repeatedly washed after sedimentation or centrifugation, collects thallus.Then contain according to 70%
Water rate mixes thallus with the nutrient solution of preparation, 2.5% preserving agent dimethyl sulfoxide is added after shaking up, in -70 DEG C of ultralow temperature
Storage in refrigerator.The activity and survival rate for investigating thallus are taken out after 5 years, by the recovery of 4d, the activity of thallus can be restored completely, deposit
Motility rate is up to 92%, and nitrosoation rate is up to 80% or more.
Concentration of the nutriment in final mixture are as follows: the NH of 0.8g/L4 +The Fe of-N, 0.03g/L2+, the Mg of 0.2g/L2 +, the K of 0.3g/L+, the Ca of 0.05g/L2+, adjusting pH is 9.0.
Embodiment 4
Processing mode and operating condition with embodiment 1, the difference is that: according to promoter concentration in cultivating system
Growth promoter III is added in 20mg/L.When the ammonia nitrogen removal frank of thallus is up to 99% or more, stop culture, training when nitrosoation rate is up to 80%
Supporting the time is 3 days, is repeatedly washed after sedimentation or centrifugation, collects thallus.Then according to 50% moisture content, by thallus with match
The preservation nutrient solution of system mixes, and 1.5% preserving agent dimethyl sulfoxide is added after shaking up, in -50 DEG C of ultra low temperature freezer preservations.3 years
Take out the activity and survival rate for investigating thallus afterwards, by the recovery of 2d, the activity of thallus can be restored completely, survival rate up to 95%,
Nitrosoation rate is up to 85% or more.
Comparative example 1
Processing mode and operating condition with embodiment 1, the difference is that: nitrite bacteria cultivate when do not add growth promote
Into agent.Culture needs 13 days when nitrosoation rate is up to 80% to ammonia nitrogen removal frank up to 99% or more.The work for investigating thallus is taken out after 3 years
Property and survival rate, by the recovery of 5d, the activity of thallus can be restored completely, and survival rate is up to 90%, and nitrosoation rate is less than 70%.
Comparative example 2
Processing mode and operating condition with embodiment 2, the difference is that: nitrite bacteria cultivate when do not add growth promote
Into agent.Culture needs 15 days when nitrosoation rate is up to 80% to ammonia nitrogen removal frank up to 99% or more.The work for investigating thallus is taken out after 4 years
Property and survival rate, by the recovery of 10d, the activity of thallus can be restored completely, and survival rate is up to 90%, and nitrosoation rate is less than 65%.
Claims (9)
1. a kind of method for preserving of nitrite bacteria, it is characterised in that include the following steps:
(1) growth promoter is added in nitrite bacteria incubation, the growth promoter includes metal salt, more amines
Matter, organic acid azanol and Na2SO3, the metal salt includes calcium salt, mantoquita, magnesium salts and/or ferrous salt；The polyamines
For the mixture of spermine, spermidine or both；The organic acid azanol is the mixing of formic acid azanol, hydroxylamine acetate or both
Object；Culture to ammonia nitrogen removal frank up to 99%, nitrosoation rate up to 80% when, collect thallus；
(2) nitrite bacteria preservation nutrient solution is prepared；
(3) nitrite bacteria that step (1) is collected is mixed with the preservation nutrient solution that step (2) is prepared, control pH is 7.5-
9.0, moisture content 40%-80%；
(4) preserving agent is added；
(5) cryogenic freezing preservation.
2. according to the method described in claim 1, it is characterized by: between the culture use of nitrite bacteria described in step (1)
Formula of having a rest activated sludge process；Nitrite bacteria is cultivated using the culture solution of human configuration or using nitrogen-containing wastewater；Training
Nutrient solution ingredient is mainly ammonium salt, ammonia nitrogen concentration 200-1500mg/L.
3. method according to claim 1 or 2, it is characterised in that: the culture pH value of nitrite bacteria described in step (1)
For 7.0-8.5, temperature is 30-40 DEG C, dissolved oxygen concentration 1-5mg/L, and incubation time is 1-10 days；After culture, pass through
Sedimentation, filtering or centrifugal method collect the thallus obtained.
4. according to the method described in claim 1, it is characterized by: metal salt is 40- in step (1) described growth promoter
100 parts by weight, polyamines are 5-30 parts by weight, and organic acid azanol is 0.05-1.5 parts by weight, Na2SO3For 10-40 weight
Part.
5. method according to claim 1 or 4, it is characterised in that: metal salt is calcium in step (1) described growth promoter
Salt, magnesium salts and mantoquita, wherein Ca2+、Mg2+And Cu2+Molar ratio be (5-15): (5-25): (0.5-5)；Either calcium salt, Asia
Molysite and mantoquita, wherein Ca2+、Fe2+And Cu2+Molar ratio be (5-15): (1-8): (0.5-5)；Either calcium salt, magnesium salts, Asia
Molysite and mantoquita, wherein Ca2+、Mg2+、Fe2+And Cu2+Molar ratio be (5-15): (5-25): (1-8): (0.5-5)；Described
Calcium salt is CaSO4Or CaCl2；Magnesium salts is MgSO4Or MgCl2；Ferrous salt is FeSO4Or FeCl2；Mantoquita is CuSO4Or
Person CuCl2。
6. according to the method described in claim 1, it is characterized by: step (1) the growth promoter additional amount is according to culture
Promoter concentration 10-50mg/L is added in system.
7. according to the method described in claim 1, it is characterized by: nitrite bacteria mixes with preservation nutrient solution in step (3)
Afterwards, pH is adjusted using sodium bicarbonate, sodium carbonate or sodium hydroxide.
8. according to the method described in claim 1, it is characterized by: step (3) nitrite bacteria and preservation nutrient solution are mixed
It closes in object, NH4 +- N concentration is 0.1-1.5g/L, Fe2+Concentration is 0.01-0.03g/L, K+Concentration is 0.05-0.5g/L, Ca2+
Concentration is 0.01-0.05g/L, Mg2+Concentration is 0.05-0.25g/L.
9. according to the method described in claim 1, using it is characterized by: preserving agent described in step (4) is dimethyl sulfoxide
Amount is the 0.5%-3% of nitrobacteria and preservation nutrient mix volume；Cryogenic freezing preservation temperature is -20~-70 DEG C.
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| CN108486019B (en) * | 2018-05-09 | 2020-05-12 | 浙江省农业科学院 | Method for preserving nitrosobacteria |
| CN114684925B (en) * | 2020-12-30 | 2023-03-24 | 中国石油化工股份有限公司 | Short-cut nitrification treatment method for ammonia-containing wastewater |
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| CN102417238A (en) * | 2011-10-18 | 2012-04-18 | 大连理工大学 | Culture method of short-cut nitrification and denitrification granular sludge |
| CN103773685A (en) * | 2012-10-23 | 2014-05-07 | 中国石油化工股份有限公司 | Method for preserving nitrosomas |
| CN105621625A (en) * | 2014-10-28 | 2016-06-01 | 中国石油化工股份有限公司 | Nitrite bacteria growth promoter and preparation method thereof |
| CN105621611A (en) * | 2014-10-28 | 2016-06-01 | 中国石油化工股份有限公司 | Method for quickly starting short-cut nitrification and denitrification of ammonia-containing wastewater |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102417238A (en) * | 2011-10-18 | 2012-04-18 | 大连理工大学 | Culture method of short-cut nitrification and denitrification granular sludge |
| CN103773685A (en) * | 2012-10-23 | 2014-05-07 | 中国石油化工股份有限公司 | Method for preserving nitrosomas |
| CN105621625A (en) * | 2014-10-28 | 2016-06-01 | 中国石油化工股份有限公司 | Nitrite bacteria growth promoter and preparation method thereof |
| CN105621611A (en) * | 2014-10-28 | 2016-06-01 | 中国石油化工股份有限公司 | Method for quickly starting short-cut nitrification and denitrification of ammonia-containing wastewater |
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