CN106554921A - A kind of method for preserving of nitrobacteria - Google Patents
A kind of method for preserving of nitrobacteria Download PDFInfo
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- CN106554921A CN106554921A CN201510635129.4A CN201510635129A CN106554921A CN 106554921 A CN106554921 A CN 106554921A CN 201510635129 A CN201510635129 A CN 201510635129A CN 106554921 A CN106554921 A CN 106554921A
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Abstract
The invention discloses a kind of method for preserving of nitrobacteria, including（1）Growth promoter is added in nitrobacteria incubation, the growth promoter includes slaine and polyamines, and wherein slaine is 40-100 weight portions, and polyamines are 5-30 weight portions；The slaine is made up of calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to entrance growth stable phase, collects thalline；（2）Prepare nitrobacteria preservation nutritional solution；（3）By step（1）The nitrobacteria and step of collection（2）The preservation nutritional solution mixing of preparation, controls moisture content for 40%-80%；（4）Addition preserving agent；（5）Freezing preservation.The present invention adds growth promoter in nitrobacteria incubation, can make thalline fast breeding, shortens incubation time, can particularly improve microbial activity and low temperature tolerance ability, being capable of quick activity recovery Jing after low temperature cold cold storage.
Description
Technical field
The present invention relates to a kind of thalline method for preserving, and in particular to a kind of method of freezing preservation nitrobacteria.
Background technology
Strain is one of main living resources, after an excellent strain is selected, it is necessary to keeps its merit constant or slack-off change few as much as possible, is just unlikely to reduce thalline performance, can apply aborning for a long time.Culture collection process has a lot, and freezen protective is a kind of effective way for solving germplasm degeneration and preventing nature accumulation property mutation, but traditional cryopreservation needs expensive programmed cooling instrument, complex steps.Vitrification completely is a kind of new method of cryopreservation; i.e. under high concentration protective agent; cell all enters glassy state in fast cooling together with protective agent; intracellular ice crystal is avoided to be formed; organ and tissue each several part is made all to enter identical state; have the advantages that equipment is simple than other Cryopreservations, the simple and frozen effect of program it is good, there is in terms of the structural intergrity for preserving organ and tissue distinctive feature.
The ultimate principle of Microbiological Culture Collection is mainly according to microbial physiology Biochemical Characteristics, artificial creation's condition, make microbial metabolism in torpescence, the downtrod resting state of growth and breeding, that is, take the conditions such as low temperature, drying, anoxia, make strain temporarily in a dormant state.A kind of good method for preserving should be able to keep the original merit of strain constant first for a long time, while it is also necessary to take into account that method itself easily and economically, to promote the use of on producing.Therefore, it is determined that on the premise of suitable method for preserving, how to improve the cold tolerance of thalline, for maintaining the premium properties of thalline, prevent spawn degeneration significant.
CN201110353746.7 discloses a kind of nitrifier method for preserving, including：（1）Will nitrification yeast culture to growing stable phase, and collect nitrobacteria；（2）Prepare nitrobacteria preservation nutritional solution；（3）Step（1）The nitrobacteria and step of collection（2）The nitrobacteria preservation nutritional solution mixing of preparation, moisture content is 40%-80%, and moisture content refers to thalline weight in wet base and nutrient solution volume ratio；（4）Addition preserving agent；（5）Freezing.The method can extend the holding time of strain, while making the activity of strain keep relative stablizing.But, as nitrobacteria growth is slower, needing will be longer in the cycle of spawn culture to stable phase.Additionally, thalline is in cryopreservation procedures, need to add high concentration nutritional material and a large amount of preserving agents, preservation is relatively costly.
The content of the invention
For the deficiencies in the prior art, the invention provides a kind of method for preserving of nitrobacteria.The present invention adds growth promoter in nitrobacteria incubation, can make thalline fast breeding, shortens incubation time, can particularly improve microbial activity and low temperature tolerance ability, Jing after low temperature cold cold storage can quick activity recovery, preservation cost is relatively low.
The method for preserving of nitrobacteria of the present invention, comprises the steps：
（1）Growth promoter is added in nitrobacteria incubation, the growth promoter includes slaine and polyamines, and wherein slaine is 40-100 weight portions, preferably 50-80 weight portions, and polyamines are 5-30 weight portions, preferably 10-20 weight portions；The slaine is made up of calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to entrance growth stable phase, collects thalline；
（2）Prepare nitrobacteria preservation nutritional solution；
（3）By step（1）The nitrobacteria and step of collection（2）The preservation nutritional solution mixing of preparation, controls moisture content for 40%-80%, preferably 50%-70%；
（4）Addition preserving agent；
（5）Freezing preservation.
Step of the present invention（1）The culture of described nitrobacteria such as adopts intermittent activated sludge process etc. using the conventional method in this area.The culture fluid of human configuration can be adopted, it would however also be possible to employ nitrogen-containing wastewater is cultivated to nitrobacteria, and culture fluid ingredient is mainly ammonium salt, and ammonia nitrogen concentration is 200-1500mg/L.The primary source of nitrobacteria culture can be the nitrobacteria for arbitrarily needing preservation.The condition of culture of nitrobacteria is：PH value is 7.0-8.5, preferably 7.5-8.0；Temperature is 20-40 DEG C, preferred 25-37 DEG C；Dissolved oxygen concentration is 1-5mg/L, preferably 2-4mg/L.Nitrobacteria culture collects the nitrobacteria that obtain by methods such as sedimentation, filtration, centrifugations to stable phase is had just enter into.
Step of the present invention（1）Slaine in the growth promoter can be calcium salt, magnesium salt and mantoquita, wherein Ca2+、Mg2+And Cu2+Mol ratio be（5-15）:（5-25）:（0.5-5）, preferably（8-12）:（10-20）:（1-4）；Or calcium salt, ferrous salt and mantoquita, wherein Ca2+、Fe2+And Cu2+Mol ratio be（5-15）:（1-8）:（0.5-5）, preferably（8-12）:（2-6）:（1-4）；Or calcium salt, magnesium salt, ferrous salt and mantoquita, wherein Ca2+、Mg2+、Fe2+And Cu2+Mol ratio be（5-15）:（5-25）:（1-8）:（0.5-5）, preferably（8-12）:（10-20）:（2-6）:（1-4）.
Step of the present invention（1）Calcium salt in the growth promoter is CaSO4Or CaCl2, preferred CaSO4；Magnesium salt is MgSO4Or MgCl2, preferred MgSO4；Ferrous salt is FeSO4Or FeCl2, preferred FeSO4；Mantoquita is CuSO4Or CuCl2, preferred CuSO4。
Step of the present invention（1）Polyamines in the growth promoter are the mixture of spermine, spermidine or both.It is preferred that a certain amount of mineral acid azanol is added in growth promoter, such as one or more in oxammonium hydrochloride., oxammonium sulfate. or phosphatic hydroxylamine, preferably oxammonium sulfate.；Addition is 0.5-15 weight portions, preferably 2-10 weight portions.The appropriate addition of mineral acid azanol can directly participate in the metabolic process of nitrobacteria, shorten enzymatic reaction process, while can be with accelerated cell growth as the activator of cell as the substrate of azanol oxygen also enzyme.
Step of the present invention（1）Described growth promoter addition is added according to promoter concentration 10-50mg/L in cultivating system, and preferred 20-30mg/L is added.
Step of the present invention（2）The ammonium salt contained in described nitrobacteria preservation nutritional solution is（NH4)2SO4, also containing trace substance FeSO4、KH2PO4、MgCl2And CaCl2.The consumption of various materials presses step（3）Needed for want concentration determine.In described nitrobacteria and preservation nutrient mix, 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 had an impact for the formation of the outer ice crystal of intracellular, and then affect preservation effect, it is therefore desirable to and suitable moisture content, it is 40%-80% that the present invention controls the moisture content of preservation system, preferably 50%-70%, moisture content refer to the weight content of water in nitrite bacteria preservation system.
Step of the present invention（4）Described preserving agent can be all conventional preserving agent of this area application, and preferred dimethyl sulfoxide, usage amount are the 0.5%-3% of nitrobacteria and preservation nutrient mix volume.Dimethyl sulfoxide is a kind of permeability protective agent, and to cytotoxic, molecular weight is little; dissolubility is good; cell can be penetrated rapidly, freezing point is reduced, permeability of the cell membrane to water is improved; delay freezing process; ICW can be made to appear before freezing extracellular, in extracellular formation ice crystal, improve intracellular electrolyte concentration; intracellular ice crystal is reduced, so that ice crystal is reduced to cell cold injury.
Step of the present invention（5）In, freezing preservation temperature is -20 DEG C -- between 70 DEG C.The freezing equipment that this area can be adopted conventional.
Compared with prior art, the invention has the advantages that：
1st, the present invention adds specific growth promoter in nitrobacteria incubation so that thalline realizes fast breeding under the collective effect of slaine, polyamines etc., shortens incubation time, solves the problems, such as that nitrobacteria increasess slowly.
2nd, adding for growth promoter can provide good nutrient environment to somatic cells, it is possible to increase the stability and freezing tolerance of institute's preservation thalline, extend the holding time of strain.Thalline Jing after low temperature cold cold storage can quick activity recovery at short notice, survival rate is high, tolerance impact capacity is strong.
3rd, growth promoter is added in nitrobacteria culture, slaine therein also acts as the effect of protective agent buffer solution while being both nutritional solution, reduces the usage amount of preserving agent, reduce the activity suppression to strain.
Specific embodiment
With reference to embodiment, the present invention is described in detail.But do not thus result in limitation of the present invention.
The ratio and formula for being first according to the growth promoter of table 1 prepares metal salt solution, polyamines and mineral acid azanol are being added in metal salt solution using front, the growth promoter I-V of different model is prepared, the growth promoter concentration is 0.5g/L.
The formula and ratio of 1 different accelerator of table
Embodiment 1
Culture fluid of the influent ammonium concentration for 300mg/L is prepared, nitrobacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 27 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 2.5-3.5mg/L.Growth promoter I is added according to promoter concentration 25mg/L in cultivating system.Stop culture after thalline is entered stablizes trophophase, incubation time is 3 days, settle or repeatedly washed after being centrifuged, collects thalline.Then according to 50% moisture content, thalline is mixed with the preservation nutritional solution prepared, 1.5% preserving agent dimethyl sulfoxide is added after shaking up, in -50 DEG C of ultra cold storage freezer preservations.The activity and survival rate for investigating thalline is taken out after 3 years, through the recovery of 2d, the activity of thalline can be recovered completely, up to 95%, ammonia nitrogen removal frank is up to more than 98% for survival rate.
Concentration of the nutrient substance in final mixture is：The NH of 0.3g/L4 +The Fe of-N, 0.01g/L2+, the Mg of 0.05g/L2+, the K of 0.1g/L+, the Ca of 0.01g/L2+。
Embodiment 2
Culture fluid of the influent ammonium concentration for 400mg/L is prepared, nitrobacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 37 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 0.5-1.5 mg/L.Growth promoter II is added according to promoter concentration 30mg/L in cultivating system.Stop culture after thalline is entered stablizes trophophase, incubation time is 4 days, settle or repeatedly washed after being centrifuged, collects thalline.Then according to 70% moisture content, thalline is mixed with the nutritional solution prepared, 2.0% preserving agent dimethyl sulfoxide is added after shaking up, in -70 DEG C of ultra cold storage freezer preservations.The activity and survival rate for investigating thalline is taken out after 5 years, through the recovery of 4d, the activity of thalline can be recovered completely, up to 90%, ammonia nitrogen removal frank is up to more than 94% for survival rate.
Concentration of the nutrient substance in final mixture is：The NH of 0.4g/L4 +The Fe of-N, 0.01g/L2+, the Mg of 0.05g/L2+, the K of 0.1g/L+, the Ca of 0.01g/L2+。
Embodiment 3
Culture fluid of the influent ammonium concentration for 600mg/L is prepared, nitrobacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 27 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 2.5-3.5 mg/L.Growth promoter III is added according to promoter concentration 20mg/L in cultivating system.Stop culture after thalline is entered stablizes trophophase, incubation time is 7 days, settle or repeatedly washed after being centrifuged, collects thalline.Then according to 60% moisture content, thalline is mixed with the nutritional solution prepared, 2.5% preserving agent dimethyl sulfoxide is added after shaking up, in -60 DEG C of ultra cold storage freezer preservations.The activity and survival rate for investigating thalline is taken out after 4 years, through the recovery of 3d, the activity of thalline can be recovered completely, up to 92%, ammonia nitrogen removal frank is up to more than 95% for survival rate.
Concentration of the nutrient substance in final mixture is：The NH of 0.6g/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+。
Embodiment 4
Culture fluid of the influent ammonium concentration for 300mg/L is prepared, nitrobacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 27 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 2.5-3.5 mg/L.Growth promoter IV is added according to promoter concentration 20mg/L in cultivating system.Stop culture after thalline is entered stablizes trophophase, incubation time is 3 days, settle or repeatedly washed after being centrifuged, collects thalline.Then according to 50% moisture content, by thalline and the nutritional solution prepared（Nutrient concentrations are same as Example 1）Mixing, adds 1.0% preserving agent dimethyl sulfoxide, in -50 DEG C of ultra cold storage freezer preservations after shaking up.The activity and survival rate for investigating thalline is taken out after 3 years, through the recovery of 2d, the activity of thalline can be recovered completely, up to 95%, ammonia nitrogen removal frank is up to more than 98% for survival rate.
Embodiment 5
Culture fluid of the influent ammonium concentration for 300mg/L is prepared, nitrobacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 27 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 2.5-3.5 mg/L.Growth promoter V is added according to promoter concentration 20mg/L in cultivating system.Stop culture after thalline is entered stablizes trophophase, incubation time is 3 days, settle or repeatedly washed after being centrifuged, collects thalline.Then according to 60% moisture content, by thalline and the nutritional solution prepared（Nutrient concentrations are same as Example 1）Mixing, adds 1.0% preserving agent dimethyl sulfoxide, in -60 DEG C of ultra cold storage freezer preservations after shaking up.The activity and survival rate for investigating thalline is taken out after 3 years, through the recovery of 2d, the activity of thalline can be recovered completely, up to 95%, ammonia nitrogen removal frank is up to more than 98% for survival rate.
Comparative example 1
, with embodiment 1, difference is for processing mode and operating condition：Growth promoter is not added during nitrobacteria culture.Cultivate to entrance and stablize trophophase needs 12 days.The activity and survival rate for investigating thalline is taken out after 3 years, through the recovery of 5d, the activity of thalline can be recovered completely, up to 90%, ammonia nitrogen removal frank is up to 95% for survival rate.
Comparative example 2
, with embodiment 2, difference is for processing mode and operating condition：Growth promoter is not added during nitrobacteria culture.Cultivate to entrance and stablize trophophase needs 15 days.The activity and survival rate for investigating thalline is taken out after 5 years, through the recovery of 10d, the activity of thalline can be recovered completely, up to 85%, ammonia nitrogen removal frank is up to 90% for survival rate.
Claims (11)
1. a kind of method for preserving of nitrobacteria, it is characterised in that comprise the steps：
（1）Growth promoter is added in nitrobacteria incubation, the growth promoter includes slaine and polyamines, and wherein slaine is 40-100 weight portions, and polyamines are 5-30 weight portions；The slaine is made up of calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to entrance growth stable phase, collects thalline；
（2）Prepare nitrobacteria preservation nutritional solution；
（3）By step（1）The nitrobacteria and step of collection（2）The preservation nutritional solution mixing of preparation, controls moisture content for 40%-80%；
（4）Addition preserving agent；
（5）Freezing preservation.
2. method according to claim 1, it is characterised in that：Step（1）The culture of described nitrobacteria adopts intermittent activated sludge process；Nitrobacteria is cultivated using the culture fluid of human configuration or using nitrogen-containing wastewater；Culture fluid ingredient is mainly ammonium salt, and ammonia nitrogen concentration is 200-1500mg/L.
3. method according to claim 1 and 2, it is characterised in that：Step（1）The culture pH value of described nitrobacteria is 7.0-8.5, and temperature is 20-40 DEG C, and dissolved oxygen concentration is 1-5mg/L, and incubation time is 1-10 days；After culture terminates, the nitrobacteria for obtaining is collected by sedimentation, filtration or centrifugal method.
4. method according to claim 1, it is characterised in that：Step（1）Slaine in the growth promoter is calcium salt, magnesium salt and mantoquita, wherein Ca2+、Mg2+And Cu2+Mol ratio be（5-15）:（5-25）:（0.5-5）；Or calcium salt, ferrous salt and mantoquita, wherein Ca2+、Fe2+And Cu2+Mol ratio be（5-15）:（1-8）:（0.5-5）；Or calcium salt, magnesium salt, ferrous salt and mantoquita, wherein Ca2+、Mg2+、Fe2+And Cu2+Mol ratio be（5-15）:（5-25）:（1-8）:（0.5-5）.
5. the method according to claim 1 or 4, it is characterised in that：Step（1）Calcium salt in the growth promoter is CaSO4Or CaCl2, magnesium salt is MgSO4Or MgCl2, ferrous salt is FeSO4Or FeCl2, mantoquita is CuSO4Or CuCl2。
6. method according to claim 1, it is characterised in that：Step（1）Polyamines in the growth promoter are the mixture of spermine, spermidine or both.
7. method according to claim 1, it is characterised in that：In step（1）Mineral acid azanol is added in described growth promoter, addition is 0.5-15 weight portions.
8. method according to claim 7, it is characterised in that：The mineral acid azanol is one or more in oxammonium hydrochloride., oxammonium sulfate. or phosphatic hydroxylamine, and addition is 2-10 weight portions.
9. method according to claim 1, it is characterised in that：Step（1）The addition of the growth promoter is added according to promoter concentration 10-50mg/L in cultivating system.
10. method according to claim 1, it is characterised in that：Step（3）In the nitrobacteria and preservation nutrient mix, 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.
11. methods according to claim 1, it is characterised in that：Step（4）Described preserving agent is dimethyl sulfoxide, and usage amount is the 0.5%-3% of nitrobacteria and preservation nutrient mix volume；Freezing preservation temperature is -20 DEG C -- 70 DEG C.
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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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
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| ENSIGN SA等: "In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper", 《J BACTERIOL》 * |
| XU GJ等: "Partial nitrification adjusted by hydroxylamine in aerobic granules under high DO and ambient temperature and subsequent Anammox for low C/N wastewater treatment", 《CHEMICAL ENGINEERING JOURNAL》 * |
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