CN106635803A - Preparation and preservation method of anammox bacterium dry microbial inoculum - Google Patents
Preparation and preservation method of anammox bacterium dry microbial inoculum Download PDFInfo
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- CN106635803A CN106635803A CN201710009335.3A CN201710009335A CN106635803A CN 106635803 A CN106635803 A CN 106635803A CN 201710009335 A CN201710009335 A CN 201710009335A CN 106635803 A CN106635803 A CN 106635803A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention relates to a preparation and preservation method of anammox bacterium dry microbial inoculum, and belongs to the technical field of nitrogen-containing wastewater biological treatment. According to the method, an anaerobic ammonium oxidation bacterium mixed culture to be preserved is taken out; centrifugation is performed; supernatant is discarded; then, 0.01M phosphate buffer solution is added; vortex uniform mixing is performed by a vortex device; centrifugation is performed for removing supernatant to obtain anaerobic ammonium oxidation bacterium precipitates; the anaerobic ammonium oxidation bacterium precipitates are mixed with a sodium alginate solution with the mass percentage of 1 to 3 percent; the nitrogen purging is performed; when the vacuum pumping pressure is lower than 25KPa, and the temperature is 50 to 70 DEG C, drying is performed; the anaerobic ammonium oxidation bacterium dry powder microbial inoculum is obtained; after the drying is completed, the anaerobic ammonium oxidation bacterium dry powder microbial inoculum is subjected to vacuum opening sealing, and is put into a 4 DEG C environment to be stored in a dark place for use. The sodium alginate is used as a drying protection agent, so that the cell damage of the anaerobic ammonium oxidation bacteria in the environment being 50 to 70 DEG C is reduced; the death rate is effectively reduced; the test result of a flow cytometry shows that the several rate is 95 percent or higher in the early storage period.
Description
Technical field
The invention discloses a kind of anaerobic ammonia oxidizing bacteria dry bacteria prepares the method with preservation, belong to nitrogenous effluent biological
Processing technology field, is related specifically to a kind of protective agent for providing protection mechanism for bacterial classification under high temperature drying environment.
Background technology
Anammox is with NH4 +- N is electron donor, NO2 -- N is that electron acceptor generates N2Microbial reaction.The skill
Art is not required to organic carbon source compared with traditional nitration denitrification denitrogenation technology, saves carbon source 100%, saves 63% aeration rate, remains
Remaining sludge yield reduces 80%, is not required to additionally add basicity, it is a kind of continuable bio-denitrifying sewage to be known as both at home and abroad
Technology.But because of its doubling time length (about 10.5 days), increases slowly, purebred culture is difficult to, and causes to be applied to actual waste water
Process and start excessive cycle, the first in the world industrialization anaerobic ammonia oxidation process processes 3.5 years as long as ammonia nitrogen waste water device used time,
Therefore culture preservation anaerobic ammonia oxidizing bacteria, sets up bacterial classification bank, and the bacterial classification for providing q.s for sewage disposal application becomes industry
Change the key of sizable application[1].[1] Chu Meihong, Ji Yuxin, the little green grass or young crops of leaf etc. the low-temperature preservation condition of Anammox bacterial classification. Hangzhoupro
State normal university journal (natural science edition) .2014 (02):164-167.
The method of culture presevation is a lot, common are atoleine method, sandy soil preserving process, liquid nitrogen method, refrigeration, freezing
Method, desivac and gel embedding method etc..Its principle is artificially created low temperature, is done mainly according to the Physiology and biochemistry feature of microorganism
Dry or anoxia condition, suppresses the metabolism of microorganism so as to which vital movement minimizes degree or in a dormant state, makes bacterium
Strain is seldom undergone mutation, to reach the purpose for keeping purebred[2].[2] Fu Meihong, Wang Yaoyao, Zhu Yanyan etc. microorganism fungus kind
Method for preserving. Hebei chemical industry .2010 (4)：33-34.
Currently, the method for Anammox bacterial classification preservation concentrates on refrigeration, freezing and desivac, while adding low temperature
Protective agent (such as dimethyl sulfoxide, trehalose, glycerine, glucose, polyvinyl alcohol, sodium alginate), preservation matrix (NH4 +-N、
NO2 -- N etc.) or buffer solution (molybdate, NO3 -- N etc.), experiment proves to play Anammox bacterial classification preservation certain positive
Effect.But refrigeration preservation effect is poor, the thalline death rate is higher；Freezing, desivac equipment cost are high and cumbersome, together
When be unfavorable for large-scale industrialization application.And high-temperature oven drying method can save equipment and time cost, at the same time facilitate bacterial classification
Transport, but there is no pertinent literature to report at present.
The anaerobic ammonia oxidizing bacteria person's character harsh to environmental condition determines that needs seek a kind of long-range efficient method for preserving.
When dry method preservation is carried out, baking temperature, drying time, protective agent, preservation temperature and activation recovering measure need to be considered to anaerobism
The impact of ammonia oxidation bacteria preservation effect.
The content of the invention
Deficiency of the present invention for current Anammox preservation technology, it is proposed that a kind of vacuum drying method carries out anaerobism ammonia
The optimization method of oxidation bacteria dry method preservation.The method is easy to operate, take it is short, can realize anaerobic ammonia oxidizing bacteria short term storage and
The target of industrial applications.
Technical scheme：
A kind of anaerobic ammonia oxidizing bacteria dry bacteria prepares the method with preservation, and step is as follows：
(1) the mixed training thing of the anaerobic ammonia oxidizing bacteria for treating preservation is taken out, centrifugation, abandoning supernatant；Add 0.01M phosphate
Cushioning liquid, vortice are vortexed and mix, and centrifugation removes supernatant, in triplicate, obtains anaerobic ammonia oxidizing bacteria sediment；
(2) the anaerobic ammonia oxidizing bacteria sediment for obtaining step (1) and the sodium alginate soln that mass fraction is 1～3%
According to mass volume ratio 5:8 mixing, mixed process carries out nitrogen purging；
(3) mixture that step (2) is obtained is placed in vacuum drying chamber, is evacuated to pressure to below 25KPa, temperature
For 50～70 DEG C, drying obtains the anaerobic ammonia oxidizing bacteria dry bacteria that moisture content is 15～30%；
(4) after drying terminates, anaerobic ammonia oxidizing bacteria dry bacteria is carried out into vacuum seal, is placed in lucifuge under 4 DEG C of environment and protects
Deposit standby.
The mixed process lucifuge of step (2) is processed.
Beneficial effects of the present invention：
1. sodium alginate reduces cell damage of the anaerobic ammonia oxidizing bacteria under 50～70 DEG C of environment as drying protective agent,
Effectively reduce its death rate, flow cytometer test result show its survival rate the preservation initial stage (10～15 days) 95% with
On；
2. longer, the generally 24～48h that carries out drying time when prepared by dry powder using desivac, and the method is in optimum temperature
Spend only 5～7h of lower drying time.
3. when being tested in the dry powder input reactor after preserving month, tie in the continuous experiments of 30 days by a definite date
Shu Shi, 0% that ratio anaerobic ammoxidation activity (SAA) of anaerobic ammonia oxidation reactor can be when starting brings up to 40%, compared to
For fresh bacterium solution.For the starting time of 30 days, activation recovering effect has reached preferable effect for this.
Description of the drawings
Fig. 1 is the drying time and survival rate of the lower bacterial classification of different drying temperatures, and wherein sodium alginate concentration is 2% (matter
Amount fraction), the use of instrument is flow cytometer.
Fig. 2 is 2% sodium alginate as the nitrogen concentration variation diagram of protectant continuous experiment three, and wherein drying temperature is 60
℃。
Fig. 3 is 2% sodium alginate as protectant continuous experiment NO2 --Nr/NH4 +-NrStoichiometric proportion variation diagram, its
Middle drying temperature is 60 DEG C.
Fig. 4 is 2% sodium alginate as protectant continuous experiment NO3 --Np/NH4 +-NrStoichiometric proportion variation diagram, its
Middle drying temperature is 60 DEG C.
Specific embodiment
Outstanding feature of the invention and marked improvement are further elucidated with reference to embodiment, is only that and is illustrated this
It is bright and be in no way limited to following instance.Anaerobic ammonia oxidizing bacteria dry bacteria is prepared using the present invention and its preservation effect is surveyed
Examination.
Embodiment 1：The drying time and survival rate of bacterial classification under different drying temperatures
Reaction condition：At different temperatures (40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C) are obtained anaerobic ammonia oxidizing bacteria
And record drying time, the flow cytometer detection survival rate after 4 DEG C of lucifuge preservations 10 days.
As a result as shown in figure 1, with the rising of baking temperature, drying time is reduced to 4h from 20h；At the same time survival rate
Also it is on a declining curve, drop to 95.5% from 99.8%, but there is extreme point at 60 DEG C.
On the whole, sodium alginate is dried in high temperature environments for anaerobic ammonia oxidizing bacteria as protective agent and played well
Protective effect.Survival rate highest is dried at 40 DEG C, but drying time is long.In view of change temperature to bacterial classification survival rate shadow
Ring little, be that its industrial applications should as far as possible shorten drying time, therefore choose 60 DEG C as temperature optimization condition.
Embodiment 2：Dry powder rejuvenation active testing
Reaction condition：The preservation of obtained dry powder vacuum sealing lucifuge will take out after one month under the conditions of 60 DEG C, use phosphoric acid
Salt buffer solution is cleaned and put into 250ml analogue reactors after sodium alginate residual.Reactor is carried out using magnetic stirring apparatus
Stirring, daily inflated with nitrogen ensures anaerobic environment to remove the oxygen entered in reactor, and gobo lucifuge is used in outside.
The nitrogen concentration of Inlet and outlet water three change as shown in Fig. 2 before the reaction 3 days be thalline hydrolyze the phase, be mainly characterized by part bacterium
Body self-dissolving, causes water outlet ammonia nitrogen concentration to be higher than influent ammonium concentration.This is the input due to there is part thalline dead during preservation
Part ammonia nitrogen is discharged after in water.And now water outlet nitrite nitrogen and nitrate nitrogen concentration close to 0, therefore, the anaerobism ammonia in the stage
Oxidation is very faint, and by the use of nitrous acid and nitric acid as the anti-nitration reaction of electron acceptor that the two is depleted, accounts for
Leading position.
In reaction the 4-15 days is the active lag phase, is mainly characterized by thalline and tapers off self-dissolving, ammonia nitrogen and nitrite nitrogen
Start to disappear simultaneously, Anammox reaction is just aobvious.In the stage, ammonia nitrogen removal frank rises to 45% from 10%, and nitrite nitrogen goes
Except rate present fall before after the trend that rises, and nitrate nitrogen has been generated.Can be obtained by Fig. 3 and Fig. 4, in this stage NO2 --Nr/NH4 +-NrAnd NO3 --Np/NH4 +-NrStill deviation theory value 1.32 and 0.26.In active lag stage, be not suitable for the thalline of inorganic environment
Reactor is washed out with water outlet, the ammonia nitrogen and content of organics increased by thalline self-dissolving is gradually reduced so that denitrification weakens.
Phase upon actuation, ammonia nitrogen removal frank and nitrous nitrogen removal efficiency are stepped up, the final clearance close 90% of the two.
And by the visible N of Fig. 3 and Fig. 4r/NH4 +-NrAnd NO3 --Np/NH4 +-NrRespectively (0.96 ± 0.02) and (0.24 ± 0.01).The latter
Close theoretical value, and the former still has certain gap with theoretical value 1.32, illustrates that denitrification is yet suffered from, but anaerobism ammonia oxygen
The ratio of change effect is gradually increasing, and occupies leading position, and this also imply that and successfully carried out rejuvenation to preservation of bacteria strain.
In addition, the present invention is to the ratio Anammox of anaerobic ammonia oxidizing bacteria dry powder is lived before reactor start-up and at the end of startup
Property SAA is determined.As a result show, after reactor runs one month, the SAA of anaerobic ammonia oxidizing bacteria is by 0kgNkgVS-1·d-1Rise to (0.27 ± 0.04) kgNkgVS-1·d-1, and the SAA measured by fresh bacterium solution is 0.68 ± 0.03) kgN
kgVS-1·d-1, activity has returned to original 40% after starting 30 days, illustrates as protective agent and to carry out sodium alginate
4 DEG C of constant-temperature preservings of vacuum drying are a kind of innovation of anaerobic ammonia oxidizing bacteria dry method preservation and effective method.
Claims (2)
1. a kind of anaerobic ammonia oxidizing bacteria dry bacteria prepares the method with preservation, it is characterised in that step is as follows：
(1) the mixed training thing of the anaerobic ammonia oxidizing bacteria for treating preservation is taken out, centrifugation, abandoning supernatant；Add 0.01M phosphate-buffereds
Solution, vortice are vortexed and mix, and centrifugation removes supernatant, in triplicate, obtains anaerobic ammonia oxidizing bacteria sediment；
(2) the anaerobic ammonia oxidizing bacteria sediment for obtaining step (1) and mass fraction be 1～3% sodium alginate soln according to
Mass volume ratio 5:8 mixing, mixed process carries out nitrogen purging；
(3) mixture that step (2) is obtained is placed in vacuum drying chamber, is evacuated to pressure to below 25KPa, temperature is 50
～70 DEG C, drying obtains the anaerobic ammonia oxidizing bacteria dry bacteria that moisture content is 15～30%；
(4) drying terminate after, anaerobic ammonia oxidizing bacteria dry bacteria is carried out into vacuum seal, be placed under 4 DEG C of environment keep in dark place it is standby
With.
2. method according to claim 1, it is characterised in that the mixed process lucifuge of step (2) is processed.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557302A (en) * | 2017-10-23 | 2018-01-09 | 大连理工大学 | A kind of lyophil preservation of anaerobic ammonia oxidizing bacteria and the method for rejuvenation |
| WO2020057332A1 (en) * | 2018-09-18 | 2020-03-26 | 江南大学 | Method for determining optimal storage temperature of anammox biofilm for wastewater treatment |
| CN112678947A (en) * | 2019-10-17 | 2021-04-20 | 江南大学 | Method for determining optimum preservation temperature of anaerobic granular sludge |
| 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 |
| CN113122465A (en) * | 2019-12-31 | 2021-07-16 | 中国石油化工股份有限公司 | Preparation and preservation method of nitrobacteria dry powder microbial inoculum |
| CN113564096A (en) * | 2021-08-30 | 2021-10-29 | 黑龙江红菌生物科技有限公司 | Preparation method of anaerobic ammonium oxidation bacterium dry powder microbial inoculum |
| CN114684927A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Method for quickly starting denitrification function of sewage treatment system |
| CN114686377A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Preparation and preservation method of nitrifying bacteria agent |
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| CN103787561A (en) * | 2014-01-23 | 2014-05-14 | 杭州师范大学 | Application of trehalose in preservation of anaerobic ammonia oxidation granular sludge |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557302A (en) * | 2017-10-23 | 2018-01-09 | 大连理工大学 | A kind of lyophil preservation of anaerobic ammonia oxidizing bacteria and the method for rejuvenation |
| WO2020057332A1 (en) * | 2018-09-18 | 2020-03-26 | 江南大学 | Method for determining optimal storage temperature of anammox biofilm for wastewater treatment |
| US11513053B2 (en) | 2018-09-18 | 2022-11-29 | Jiangnan University | Method for determining optimal preservation temperature of anaerobic ammonium oxidation biofilm in wastewater treatment for total nitrogen removal |
| CN112678947A (en) * | 2019-10-17 | 2021-04-20 | 江南大学 | Method for determining optimum preservation temperature of anaerobic granular sludge |
| 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 |
| CN113122465A (en) * | 2019-12-31 | 2021-07-16 | 中国石油化工股份有限公司 | Preparation and preservation method of nitrobacteria dry powder microbial inoculum |
| CN114684927A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Method for quickly starting denitrification function of sewage treatment system |
| CN114686377A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Preparation and preservation method of nitrifying bacteria agent |
| CN114686377B (en) * | 2020-12-31 | 2023-07-04 | 中国石油化工股份有限公司 | Preparation and preservation method of nitrifying bacteria agent |
| CN114684927B (en) * | 2020-12-31 | 2023-09-01 | 中国石油化工股份有限公司 | Method for quickly starting denitrification function of sewage treatment system |
| CN113564096A (en) * | 2021-08-30 | 2021-10-29 | 黑龙江红菌生物科技有限公司 | Preparation method of anaerobic ammonium oxidation bacterium dry powder microbial inoculum |
| CN113564096B (en) * | 2021-08-30 | 2024-02-02 | 黑龙江红菌生物科技有限公司 | Preparation method of anaerobic ammonia oxidation bacteria dry powder microbial inoculum |
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Application publication date: 20170510 |