CN106554922A - 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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- CN106554922A CN106554922A CN201510635144.9A CN201510635144A CN106554922A CN 106554922 A CN106554922 A CN 106554922A CN 201510635144 A CN201510635144 A CN 201510635144A CN 106554922 A CN106554922 A CN 106554922A
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Abstract
The invention discloses a kind of method for preserving of nitrite bacteria, including（1）Growth promoter is added in nitrite bacteria incubation, the accelerator includes slaine, polyamines, organic acid azanol and Na2SO3, the slaine includes calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to ammonia nitrogen removal frank up to 99%, nitrosoation rate up to 80% when, collects thalline；（2）Prepare nitrite bacteria preservation nutritional solution；（3）By step（1）The nitrite bacteria and step of collection（2）The preservation nutritional solution mixing of preparation, it is 7.5-9.0 to control pH, and moisture content is 40%-80%；（4）Addition preserving agent；（5）Freezing preservation.The present invention adds growth promoter in nitrite bacteria incubation, can make thalline fast breeding, improves nitrosoation rate, shortens incubation time；Microbial activity and low temperature tolerance ability can be particularly improved, 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 nitrite bacteria.
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.
It is nitrate nitrogen or nitrite nitrogen by ammonium oxidation by Nitromonas or nitrite bacteria that the nitrifying process of biological denitrificaion is under aerobic condition, then nitrate nitrogen or nitrite nitrogen are reduced into gaseous nitrogen by the denitrifying bacterium under anoxia condition remove from water.One is wasted in the middle of traditional biological denitrification process by nitrous nitrogen transformation nitrate nitrogen, nitrate nitrogen is converted into the process of nitrite nitrogen again.Short-cut nitrification and denitrification denitrogenation is to control the further nitrification of nitrite nitrogen is prevented in the nitrite nitrogen stage by nitration reaction, then directly carries out denitrification.Compared with traditional nitration denitrification, short-cut nitrification and denitrification has the following advantages：(1) the nitrification stage can reduce by 25% or so oxygen demand, reduce energy consumption；(2) the denitrification stage can reduce by 40% or so organic carbon source, reduce operating cost；(3) shortening is asked when reacting, reactor volume can reduce 30%-40% or so；(4) with higher denitrification rate；(5) sludge yield is reduced；(6) reduce throwing alkali number etc..As can be seen here, control nitrifying process significant in Nitrification Stage, and after the high-performance nitrite bacteria for completing short distance nitration is selected, how permanently effective preservation is most important.
CN201210404073.8 discloses a kind of method for preserving of nitrite bacteria, including：（（1）By nitrite bacteria culture to nitrosoation rate up to more than 80%, and collects thalline；（2）Prepare nitrite bacteria preservation nutritional solution；（3）Step（1）The nitrite bacteria body of collection and step（2）The nitrite bacteria preservation nutritional solution mixing of preparation, pH is 7.5-9.0, and moisture content is 40%-80%, and moisture content refers to the weight content of water in nitrite bacteria preservation system；（4）Addition preserving agent；（5）Freezing preservation.The method can extend the holding time of strain, while making the activity of strain keep relative stablizing.But as nitrite bacteria grows relatively slowly, needing will be spawn culture longer to the cycle for growing 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 nitrite bacteria.The present invention adds growth promoter in nitrite bacteria incubation, can make thalline fast breeding, improves nitrosoation rate, shortens incubation time；Microbial activity and low temperature tolerance ability can particularly be improved, Jing after low temperature cold cold storage can quick activity recovery, preservation cost is relatively low.
The method for preserving of nitrite bacteria of the present invention, comprises the steps：
（1）Growth promoter is added in nitrite bacteria incubation, the growth promoter includes slaine, polyamines, organic acid azanol and Na2SO3, the slaine includes calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to ammonia nitrogen removal frank up to 99%, nitrosoation rate up to more than 80% when, collects thalline；
（2）Prepare nitrite bacteria preservation nutritional solution；
（3）By step（1）The nitrite bacteria and step of collection（2）The preservation nutritional solution mixing of preparation, it is 7.5-9.0 to control pH, and moisture content is 40%-80%, preferably 50%-70%；
（4）Addition preserving agent；
（5）Freezing preservation.
Step of the present invention（1）The culture of described nitrite bacteria 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 nitrite bacteria, and culture fluid ingredient is mainly ammonium salt, and ammonia nitrogen concentration is 200-1500mg/L.The primary source of nitrite bacteria culture can be the nitrite bacteria for arbitrarily needing preservation.The condition of culture of nitrite bacteria is：PH value is 7.0-8.5, preferably 8.0-8.5；Temperature is 30-40 DEG C, preferred 35-38 DEG C；Dissolved oxygen concentration is 1-5mg/L, preferably 2-3mg/L.When culture is to ammonia nitrogen removal frank up to 99%, nitrosoation rate（Nitrosoation rate refers to the ratio that mineralized nitrogen is nitrite nitrogen）During up to 80%, nitrous acid thalline is collected by methods such as sedimentation, filtration, centrifugations.
Step of the present invention（1）Slaine in the growth promoter is 40-100 weight portions, preferably 50-80 weight portions, and polyamines are 5-30 weight portions, preferably 10-20 weight portions, and organic acid azanol is 0.05-1.5 weight portions, preferably 0.1-1.0 weight portions, Na2SO3For 10-40 weight portions, preferably 20-30 weight portions.
Step of the present invention（1）Slaine in the growth promoter includes calcium salt, mantoquita, magnesium salt and/or ferrous salt, can such as be calcium salt, ferrous salt and mantoquita, 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 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, magnesium salt, ferrous salt and mantoquita, 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 CaCl2；Magnesium salt is MgSO4Or MgCl2, preferred MgCl2；Mantoquita is CuSO4Or CuCl2, preferred CuCl2；Iron salt is FeSO4Or FeCl2, preferred FeCl2。
Step of the present invention（1）Described polyamines are the mixture of spermine, spermidine or both.Described organic acid azanol is the mixture of formic acid azanol, hydroxylamine acetate or both.
Step of the present invention（1）The addition of described growth promoter 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 nitrite bacteria 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.
Step of the present invention（3）In, after nitrite bacteria is mixed with preservation nutritional solution, pH can be adjusted using sodium bicarbonate, sodium carbonate or sodium hydroxide etc..PH is respectively provided with certain impact in the activity of Nitrification Stage and nitrite bacteria on controlling nitration reaction, therefore it is most important for the long term storage of nitrite bacteria to control the suitable pH of mixed liquor.In nitrite bacteria 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 between -20 DEG C～-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 nitrite bacteria incubation so that thalline is in slaine, polyamines, organic acid azanol and Na2SO3Collective effect under realize fast breeding, improve nitrosoation rate, shorten incubation time, solve the problems, such as that nitrite bacteria 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 nitrite bacteria 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, using front by polyamines, organic acid acid azanol and Na2SO3It is added in metal salt solution, prepares the nitrite bacteria growth promoter I-III of different model, the promoter concentration is 0.5g/L.
The formula and ratio of 1 nitrite bacteria growth promoter of table
Embodiment 1
Culture fluid of the influent ammonium concentration for 400mg/L is prepared, nitrite bacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 32 DEG C of temperature, pH is 7.8, dissolved oxygen DO is 1.0-2.5mg/L.Growth promoter I is added according to promoter concentration 25mg/L in cultivating system.When the ammonia nitrogen removal frank of thalline is up to more than 99%, nitrosoation rate is 3 days up to culture, incubation time is stopped when 80%, is repeatedly washed, collects thalline after sedimentation or centrifugation.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%, nitrosoation rate is up to more than 80% 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+, it is 8.5 to adjust pH.
Embodiment 2
Culture fluid of the influent ammonium concentration for 600mg/L is prepared, nitrite bacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 35 DEG C of temperature, pH is 8.0, dissolved oxygen DO is 1.5-3.0mg/L.Growth promoter II is added according to promoter concentration 30mg/L in cultivating system.When the ammonia nitrogen removal frank of thalline is up to more than 99%, nitrosoation rate is 5 days up to culture, incubation time is stopped when 80%, is repeatedly washed, collects thalline after sedimentation or centrifugation.Then according to 60% moisture content, thalline is mixed with the nutritional solution prepared, 2.0% 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 90%, nitrosoation rate is up to more than 80% for survival rate.
Concentration of the nutrient substance in final mixture is：The NH of 0.6g/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+, it is 8.0 to adjust pH.
Embodiment 3
Culture fluid of the influent ammonium concentration for 800mg/L is prepared, nitrite bacteria is cultivated using intermittent activated sludge process in 100L bio-aeration reaction tanks, 37 DEG C of temperature, pH is 8.5, dissolved oxygen DO is 1.5-2.5mg/L.Growth promoter III is added according to promoter concentration 20mg/L in cultivating system.When the ammonia nitrogen removal frank of thalline is up to more than 99%, nitrosoation rate is 7 days up to culture, incubation time is stopped when 80%, is repeatedly washed, collects thalline after sedimentation or centrifugation.Then according to 70% moisture content, thalline is mixed with the nutritional solution prepared, 2.5% 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 92%, nitrosoation rate is up to more than 80% for survival rate.
Concentration of the nutrient substance in final mixture is：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+, it is 9.0 to adjust pH.
Embodiment 4
, with embodiment 1, difference is for processing mode and operating condition：Growth promoter III is added according to promoter concentration 20mg/L in cultivating system.When the ammonia nitrogen removal frank of thalline is up to more than 99%, nitrosoation rate is 3 days up to culture, incubation time is stopped when 80%, is repeatedly washed, collects thalline after sedimentation or centrifugation.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%, nitrosoation rate is up to more than 85% for survival rate.
Comparative example 1
, with embodiment 1, difference is for processing mode and operating condition：Growth promoter is not added during nitrite bacteria culture.Cultivate to ammonia nitrogen removal frank up to more than 99%, nitrosoation rate needs 13 days up to when 80%.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, and, up to 90%, nitrosoation rate is less than 70% for survival rate.
Comparative example 2
, with embodiment 2, difference is for processing mode and operating condition：Growth promoter is not added during nitrite bacteria culture.Cultivate to ammonia nitrogen removal frank up to more than 99%, nitrosoation rate needs 15 days up to when 80%.The activity and survival rate for investigating thalline is taken out after 4 years, through the recovery of 10d, the activity of thalline can be recovered completely, and, up to 90%, nitrosoation rate is less than 65% for survival rate.
Claims (10)
1. a kind of method for preserving of nitrite bacteria, it is characterised in that comprise the steps：
（1）Growth promoter is added in nitrite bacteria incubation, the growth promoter includes slaine, polyamines, organic acid azanol and Na2SO3, the slaine includes calcium salt, mantoquita, magnesium salt and/or ferrous salt；Cultivate to ammonia nitrogen removal frank up to 99%, nitrosoation rate up to 80% when, collects thalline；
（2）Prepare nitrite bacteria preservation nutritional solution；
（3）By step（1）The nitrite bacteria and step of collection（2）The preservation nutritional solution mixing of preparation, it is 7.5-9.0 to control pH, and moisture content is 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 nitrite bacteria adopts intermittent activated sludge process；Nitrite bacteria 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 nitrite bacteria is 7.0-8.5, and temperature is 30-40 DEG C, and dissolved oxygen concentration is 1-5mg/L, and incubation time is 1-10 days；After culture terminates, the thalline for obtaining is collected by sedimentation, filtration or centrifugal method.
4. method according to claim 1, it is characterised in that：Step（1）In the growth promoter slaine be 40-100 weight portions, polyamines be 5-30 weight portions, organic acid azanol be 0.05-1.5 weight portions, Na2SO3For 10-40 weight portions.
5. the method according to claim 1 or 4, it is characterised in that：Step（1）In the growth promoter slaine be 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）；Described calcium salt is CaSO4Or CaCl2；Magnesium salt is MgSO4Or MgCl2；Ferrous salt is FeSO4Or FeCl2；Mantoquita is CuSO4Or CuCl2。
6. the method according to claim 1 or 4, it is characterised in that：Step（1）Polyamines in the growth promoter are the mixture of spermine, spermidine or both；The organic acid azanol is the mixture of formic acid azanol, hydroxylamine acetate or both.
7. method according to claim 1, it is characterised in that：Step（1）The growth promoter addition is added according to promoter concentration 10-50mg/L in cultivating system.
8. method according to claim 1, it is characterised in that：Step（3）After middle nitrite bacteria is mixed with preservation nutritional solution, pH is adjusted using sodium bicarbonate, sodium carbonate or sodium hydroxide.
9. method according to claim 1, it is characterised in that：Step（3）In the nitrite bacteria 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.
10. method 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～-70 DEG C.
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| CN108486019A (en) * | 2018-05-09 | 2018-09-04 | 浙江省农业科学院 | The method for preserving of nitrococcus |
| CN114684925A (en) * | 2020-12-30 | 2022-07-01 | 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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| 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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