CN108117992A - A kind of preservation under room temperature method of denitrifying bacterium - Google Patents
A kind of preservation under room temperature method of denitrifying bacterium Download PDFInfo
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Abstract
The present invention relates to a kind of preservation under room temperature method of denitrifying bacterium, including（1）By denitrifying bacterium culture to exponential phase, and collect thalline；（2）Preservation nutrient solution is prepared, mainly including carbon source, nitrogen source and micro substance etc.；（3）Step（1）Denitrifying bacterium and step（2）Preservation nutrient solution mixes in preservation container, adds in appropriate preservation auxiliary agent, and the preservation auxiliary agent includes glycolipid, sugar alcohol and acylate；（4）By step（3）Mixed liquor sealing 4 12h of culture, preservation under room temperature.Compared with prior art, the method for the present invention is suitable for the room temperature long term storage of a large amount of denitrifying bacteriums, has many advantages, such as that room temperature preservation effect is good, survival rate is high, activation recovering is fast.
Description
Technical field
The present invention relates to a kind of thalline method for preserving, and in particular to a kind of side of simple and effective preservation under room temperature denitrifying bacterium
Method.
Background technology
Ammonia nitrogen is the pollutant that country realizes water pollution control overall control, and ammonia-nitrogen content is excessively high in water body can cause water quality
Eutrophication causes some algae excessive propagations in water body, and other biological growths are affected, so as to destroy aquatic ecosystem
System causes water quality deterioration and influences it to use function.Bio-denitrification technology is with the advantages that its environment-friendly high-efficiency non-secondary pollution
The hot spot of the application of the technical research of denitrogenation both at home and abroad at present.Biological denitrificaion is first by ammonia nitrogen oxygen under aerobic condition by nitration reaction
Nitrate nitrogen or nitrite nitrogen are turned to, then nitrate nitrogen or nitrite nitrogen are reduced into from water by gaseous nitrogen by the anti-nitration reaction under anoxia condition
It removes.It can be seen that denitrification process is only the step of real denitrogenation, and denitrifying high-performance denitrifying bacterium is completed by selection and breeding
After out, how permanently effective preservation is most important.
Microorganism fungus kind is one of important living resources, after an excellent strain is selected, it is necessary to keep
Its merit is constant or lacks slack-off change as much as possible, is just unlikely to reduce thalline performance, energy prolonged application is in production.Strain
For the basic principle of preservation mainly according to its physiological and biochemical property, artificial creation's condition makes microbial metabolism be in torpescence, life
The dormant state of long reproduction inhibition takes the conditions such as low temperature, drying, anoxic, make strain temporarily in a dormant state.It is a kind of
Good method for preserving should be able to keep the original merit of strain constant for a long time first, while it is also necessary to take into account that the letter of method in itself
Just it is and economical, to be promoted the use of in production.
For effectively conserving microorganism bacterial, it is necessary to which suitable method for preserving is selected in the characteristic research for strain.
There are many culture collection process, common both at home and abroad at present to have regular grafting, atoleine method, sandy soil tube method, vacuum refrigeration to do
Dry method, ultra low temperature freezer freezing, ultra-low temperature liquid nitrogen freezing etc..Wherein regular grafting, atoleine method, sandy soil tube method,
Vacuum freeze-drying method is not suitable for the preservation of a large amount of strains；Ultra-low temperature liquid nitrogen freezing and ultra low temperature freezer freezing etc. are both needed to make
Cell suspension is prepared with protective agent, protective agent has cow's milk, serum, carbohydrate, glycerine, dimethyl sulfoxide etc., it is therefore an objective to prevent because cold
Jelly or moisture constantly damage of the distillation to cell.Freezen protective is to solve one kind that germplasm degenerates and prevents nature accumulation property mutation
Effective way, but traditional Cord blood needs expensive programmed cooling instrument device, complex steps.Complete vitrifying is Cord blood
A kind of new method, i.e., under high concentration protective agent, cell together with protective agent in fast cooling all into glassy state, keep away
Exempt from intracellular ice crystal to be formed, make organ and tissue each several part all into identical state, with equipment is simple, program is simple and freezes
The advantages that effect is good.But whether protective agent only can guarantee the survival rate of thalline, have an impact to microbial activity and effect
It is also indefinite.
CN101307293 provides a kind of culture collection process, using semisolid, the method for low temperature seal culture：By bacterium
Kind is inoculated in sterile semisolid culturemedium, is poured into after sterile liquid paraffin in the environment temperature for store vertically 6~8 DEG C and is protected
It deposits.The strain is staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, bacillus subtilis, clostridium sporogenes, black
Aspergillus or white chain pearl bacterium.The holding time of strain can be extended using the method, while the concentration of strain keeps relative stability, and is
Substantial amounts of cost has been saved in production, experiment, is reduced processing and discharge that strain uses rear discarded object, is reduced the danger to environment
Evil.But procedure complexity, be not suitable for the preservation of a large amount of thalline.
CN103773684 is related to a kind of method for preserving of denitrifying bacterium, includes the following steps：（1）By denitrifying bacterium culture
To growth stationary phase, and collect thalline；（2）Prepare denitrifying bacterium preservation nutrient solution；（3）Step（1）The denitrification thalline of collection
With step（2）The preservation nutrient solution mixing of preparation, moisture content is 40%~80%, and moisture content refers to water in denitrifying bacterium preservation system
Weight content；（4）Cryogenic freezing preservation.This method can extend the holding time of strain, and the activity of strain is made to keep relatively steady
It is fixed.But the method freezing temperature is -20~-70 DEG C, and the thalline of short term storage is needed for some, it is not economic and practical.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of preservation under room temperature method of denitrifying bacterium, it is suitable for a large amount of
Denitrifying bacterium is preserved, has many advantages, such as that room temperature preservation effect is good, survival rate is high, activation recovering is fast.
The preservation under room temperature method of denitrifying bacterium of the present invention, including following content：
（1）By denitrifying bacterium culture to exponential phase, and collect thalline；
（2）Preservation nutrient solution is prepared, mainly including carbon source, nitrogen source and micro substance etc.；
（3）Step（1）Denitrifying bacterium and step（2）Preservation nutrient solution mixes in preservation container, adds in appropriate preservation auxiliary agent, institute
Stating preservation auxiliary agent includes glycolipid, sugar alcohol and acylate；
（4）By step（3）Mixed liquor sealing culture 4-12h, preservation under room temperature.
In the present invention, step（1）The denitrifying bacterium culture method that uses this field routine, such as SBR methods.Denitrifying bacterium
The culture solution manually prepared may be employed in culture solution, denitrifying bacterium can also be cultivated using actual waste water.Denitrifying bacterium
Nitrogen source contained by culture solution is nitrate nitrogen and/or nitrite nitrogen, and nitrogen concentration is 100~800mg/L；Institute's carbonaceous sources are sweet
Oil, methanol etc., concentration are calculated as 1000~5000mg/L with COD, also containing a small amount of Fe2+、Mg2+、K+、Ca2+Wait metal ions and
Phosphate anion etc..Denitrifying bacterium condition of culture is：15~40 DEG C of temperature, pH are 7.0~8.5, and dissolved oxygen concentration is less than 1mg/
L, 10~50rpm of speed of agitator cultivate 1~5 day to exponential phase.Denitrifying bacterium culture to exponential phase, by sedimentation,
The methods of filtering, centrifugation, collects the denitrification thalline obtained.The primary source of denitrifying bacterium culture can arbitrarily need to protect
The denitrifying bacterium of Tibetan.
In the present invention, step（2）Carbon source is glycerine in preservation nutrient solution, and nitrogen source is nitrate nitrogen（NO3 --N）And/or nitrous
Hydrochlorate nitrogen（NO2 --N）, if nitrate is NaNO3、KNO3Deng nitrite NaNO2Deng micro substance FeSO4、KH2PO4、
MgCl2And CaCl2.The dosage of various substances presses step（3）Needed for concentration determine.
In the present invention, step（3）In denitrifying bacterium and preservation nutrient mix, glycerol concentration is calculated as 0.5 with COD~
3.0g/L, NO3 -- N and/or NO2 -- N concentration is 0.2~0.8g/L, Fe2+Concentration is 0.01~0.06g/L, K+Concentration is 0.05
~0.5g/L, Ca2+Concentration is 0.01~0.1g/L, Mg2+Concentration is 0.05~0.5g/L.
In the present invention, step（3）In the preservation auxiliary agent, in parts by weight, glycolipid content is 0.5-15 parts by weight, preferably
2-10 parts by weight；Content of sugar alcohol is 0.5-15 parts by weight, preferably 2-10 parts by weight；Acylate is 5-30 parts by weight, is preferably
10-20 parts by weight.The glycolipid is at least one of seaweed glycolipid, sophorolipid and rhamnolipid etc., preferably lactone type sophorose
Fat；Sugar alcohol is selected from one or more of mannitol, xylitol, lactitol, ribitol, galactitol, inositol and erythrite etc.,
It is preferred that lactitol；Acylate is one or more of sodium acetate, sodium succinate and sodium citrate etc., preferably sodium acetate.Make
Dosage is 0.01-5.0mg/L according to preservation agent concentration in preservation system, and preferably 0.5-1.0mg/L is added.
In the present invention, step（4）Seal the same step of condition of culture（1）The condition of culture of denitrifying bacterium.Preservation under room temperature temperature
It it is preferably 0 DEG C~20 DEG C for -10 DEG C~30 DEG C.
Compared with prior art, the invention has the advantages that：
（1）Complex optimum is carried out from denitrifying bacterium growth period, preservative fluid composition, preservation auxiliary agent etc. are many-sided, can realize anti-nitre
Change the long term storage under bacterium normal temperature condition, bacterial activity keeps relative stability, preservation under room temperature survival rate height, activity after 1-12 months
Recover rapid, for the very economical convenience of the short term storage of a large amount of thalline.
（2）By adding in specific preservation auxiliary agent, good thalline preservation can be realized under the conditions of -10 DEG C~30 DEG C
Performance reduces preservation energy consumption.
（3）The method of the present invention is simple, is not required to special installation, and economical and convenient is suitble to the preservation of a large amount of strains, preservation under room temperature 1
Nian Hou, survival rate can reach more than 90%.
Specific embodiment
The method of the present invention and effect are described in detail with reference to specific embodiment.
The preservation auxiliary agent that the present invention uses is prepared according to the ratio and formula of table 1.The promoter concentration is 0.5g/L.
The formula and ratio of 1 preservation auxiliary agent of table
Embodiment 1
Prepare NO2 -- N concentration is 200mg/L, and glycerine is carbon source, and concentration is calculated as the culture solution of 2500mg/L with COD, is given birth in 10L
Denitrifying bacterium is cultivated using SBR methods in object reaction tank, temperature is 27 DEG C, pH 7.8, speed of agitator 20rpm.Work as thalline
Stop culture, sedimentation, collected after centrifugation thalline into after stablizing growth period.
Preservation nutrient solution is prepared, according in denitrifying bacterium and preservation nutrient mix, nutrient concentrations are：Glycerine is dense
Degree is calculated as 1.0g/L, NO with COD2 -- N concentration is 0.2g/L, Fe2+Concentration is 0.02g/L, K+Concentration is 0.1g/L, Ca2+It is dense
It spends for 0.05g/L, Mg2+Concentration is prepared for 0.1g/L.
Thalline with the nutrient solution prepared is mixed, preservation auxiliary agent A is added for 0.5mg/L according to preservation agent concentration in system,
In 20 DEG C of preservations.The activity and survival rate for investigating thalline are taken out after 3 months, by the recovery of 6h, microbial activity can be completely extensive
Multiple, survival rate is up to 95%.
Embodiment 2
With embodiment 1, difference is for incubation and operating condition：In 0 DEG C of preservation.The activity for investigating thalline is taken out after 12 months
And survival rate, by the recovery of 10h, microbial activity can recover completely, and survival rate is up to 95%.
Embodiment 3
With embodiment 1, difference is for incubation and operating condition：According to preservation agent concentration in system guarantor is added for 1.0mg/L
Hide auxiliary agent A.The activity and survival rate for investigating thalline are taken out after 6 months, by the recovery of 8h, microbial activity can recover completely,
Survival rate is up to 95%.
Embodiment 4
With embodiment 1, difference is for incubation and operating condition：Using preservation auxiliary agent B.It is taken out after 3 months and investigates thalline
Activity and survival rate, by the recovery of 7h, microbial activity can recover completely, and survival rate is up to 94%.
Embodiment 5
With embodiment 1, difference is for incubation and operating condition：Using preservation auxiliary agent C.It is taken out after 3 months and investigates thalline
Activity and survival rate, by the recovery of 7h, microbial activity can recover completely, and survival rate is up to 92%.
Embodiment 6
It is 600mg/L to prepare nitrate nitrogen concentration, and glycerine is carbon source, and concentration is calculated as the culture solution of 4000mg/L with COD, in 10L biologies
Denitrifying bacterium is cultivated using SBR methods in reaction tank, temperature is 30 DEG C, pH 8.0, speed of agitator 40rpm.When thalline into
Stop culture, sedimentation or collected after centrifugation thalline after entering stable growth period.
Preservation nutrient solution is prepared, according in denitrifying bacterium and preservation nutrient mix, nutrient concentrations are：Glycerine is dense
Degree is calculated as 2.5g/L, NO with COD3 -- N concentration is 0.6g/L, Fe2+Concentration is 0.02g/L, K+Concentration is 0.1g/L, Ca2+It is dense
It spends for 0.05g/L, Mg2+Concentration is prepared for 0.1g/L.
Thalline with the nutrient solution prepared is mixed, preservation auxiliary agent A is added for 0.8mg/L according to preservation agent concentration in system,
In 15 DEG C of preservations.The activity and survival rate for investigating thalline are taken out after 6 months, by the recovery of 8h, microbial activity can be completely extensive
Multiple, survival rate is up to 95%.
Embodiment 7
It is 200mg/L to prepare nitrate nitrogen concentration, and nitrous nitrogen concentration is 200mg/L, and glycerine is carbon source, and concentration is calculated as 3000mg/ with COD
The culture solution of L cultivates denitrifying bacterium using SBR methods in 10L biological reaction pools, and temperature is 32 DEG C, and pH 8.0 is stirred
Mix rotating speed 30rpm.Stop culture, sedimentation or collected after centrifugation thalline after thalline enters stable growth period.
Preservation nutrient solution is prepared, according in denitrifying bacterium and preservation nutrient mix, nutrient concentrations are：Glycerine is dense
Degree is calculated as 1.5g/L, NO with COD3 -- N concentration is 0.2g/L, NO2 -- N concentration is 0.2g/L, Fe2+Concentration is 0.02g/L, K+
Concentration is 0.1g/L, Ca2+Concentration is 0.05g/L, Mg2+Concentration is prepared for 0.1g/L.
Thalline with nutrient solution is mixed, preservation auxiliary agent A is added according to preservation agent concentration in system for 0.6mg/L, in 5 DEG C of guarantors
It hides.The activity and survival rate for investigating thalline are taken out after 6 months, by the recovery of 8h, microbial activity can recover completely, survival rate
Up to 95%.
Comparative example 1
With embodiment 1, difference is for incubation and operating condition：Preservation auxiliary agent A is not used.Thalline starts to black and have after 15 days
Taste takes out the activity and survival rate for investigating thalline, and by the recovery of 12h, microbial activity recovers substantially, and survival rate is up to 80%.
Comparative example 2
With embodiment 1, difference is for incubation and operating condition：It is cultivated after adding in preserving agent without sealing.It is taken after 3 months
Go out to investigate the activity and survival rate of thalline, by the recovery of 10h, microbial activity recovers substantially, and survival rate is up to 85%.
Comparative example 3
With embodiment 1, difference is for incubation and operating condition：Using preserving agent D, E, F.It is taken out after 3 months and investigates thalline
Activity and survival rate, by the recovery of 6h, microbial activity can recover substantially, and survival rate is up to 85% or so.
Claims (10)
1. a kind of preservation under room temperature method of denitrifying bacterium, it is characterised in that comprise the following steps：
（1）By denitrifying bacterium culture to exponential phase, and collect thalline；
（2）Preservation nutrient solution is prepared, mainly including carbon source, nitrogen source and micro substance；
（3）Step（1）Denitrifying bacterium and step（2）Preservation nutrient solution mixes in preservation container, adds in appropriate preservation auxiliary agent, institute
Stating preservation auxiliary agent includes glycolipid, sugar alcohol and acylate；
（4）By step（3）Mixed liquor sealing culture 4-12h, preservation under room temperature.
2. according to the method described in claim 1, it is characterized in that：Step（3）In the preservation auxiliary agent, in parts by weight, sugar
Fat content is 0.5-15 parts by weight, and content of sugar alcohol is 0.5-15 parts by weight, and acylate is 5-30 parts by weight.
3. method according to claim 1 or 2, it is characterised in that：In preservation auxiliary agent, the glycolipid is seaweed glycolipid, Chinese scholartree
At least one of glycolipid and rhamnolipid；Sugar alcohol is selected from mannitol, xylitol, lactitol, ribitol, galactitol, inositol
One or more of with erythrite；Acylate is one or more of sodium acetate, sodium succinate and sodium citrate.
4. according to the method described in claim 3, it is characterized in that：In preservation auxiliary agent, glycolipid is lactone type sophorolipid, and sugar alcohol is
Lactitol, acylate are sodium acetate.
5. method according to claim 1 or 2, it is characterised in that：The usage amount of preservation auxiliary agent in preservation system according to protecting
Agent concentration is hidden to be added for 0.01-3.0mg/L.
6. according to the method described in claim 1, it is characterized in that：Step（1）Denitrifying bacterium culture using SBR methods, culture
Liquid is using the culture solution manually prepared or actual waste water, and nitrogen source contained by culture solution is nitrate nitrogen and/or nitrite nitrogen, nitrogen
Source concentration is 100~800mg/L；Institute's carbonaceous sources are glycerine or methanol, and concentration is calculated as 1000~5000mg/L with COD, also contains
A small amount of Fe2+、Mg2+、K+、Ca2+And phosphate anion.
7. the method according to claim 1 or 6, it is characterised in that：Step（1）Denitrifying bacterium condition of culture is：Temperature 15
~40 DEG C, pH is 7.0~8.5, and dissolved oxygen concentration is less than 1mg/L, 10~50rpm of speed of agitator, cultivates 1~5 day to logarithm life
For a long time.
8. according to the method described in claim 1, it is characterized in that：Step（2）Carbon source is glycerine in preservation nutrient solution, and nitrogen source is
Nitrate nitrogen and/or nitrite nitrogen, nitrate NaNO3And/or KNO3, nitrite NaNO2, micro substance is
FeSO4、KH2PO4、MgCl2And CaCl2。
9. according to the method described in claim 1, it is characterized in that：Step（3）In denitrifying bacterium and preservation nutrient mix,
Glycerol concentration is calculated as 0.5~3.0g/L, NO with COD3 -- N and/or NO2 -- N concentration is 0.2~0.8g/L, Fe2+Concentration is
0.01~0.06g/L, K+Concentration is 0.05~0.5g/L, Ca2+Concentration is 0.01~0.1g/L, Mg2+Concentration for 0.05~
0.5g/L。
10. according to the method described in claim 1, it is characterized in that：Step（4）Preservation under room temperature temperature is -10 DEG C~30 DEG C.
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| CN113122454A (en) * | 2019-12-31 | 2021-07-16 | 中国石油化工股份有限公司 | Dry preservation method of nitrifying bacteria |
| CN113603235A (en) * | 2021-08-18 | 2021-11-05 | 中冶生态环保集团有限公司 | Preservation method for IFAS/MBBR process curing suspension carrier |
| CN114686392A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Normal-temperature preservation method of nitrifying bacteria |
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| CN113603235A (en) * | 2021-08-18 | 2021-11-05 | 中冶生态环保集团有限公司 | Preservation method for IFAS/MBBR process curing suspension carrier |
| CN115353197A (en) * | 2022-06-27 | 2022-11-18 | 临清三和纺织集团有限公司 | Denitrification treatment process for removing total nitrogen in sewage |
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| CN108117992B (en) | 2021-05-04 |
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