CN104560821B - A kind of Nitrobacter winogradskyi XY 01 and its cultural method and application - Google Patents
A kind of Nitrobacter winogradskyi XY 01 and its cultural method and application Download PDFInfo
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- CN104560821B CN104560821B CN201410852449.0A CN201410852449A CN104560821B CN 104560821 B CN104560821 B CN 104560821B CN 201410852449 A CN201410852449 A CN 201410852449A CN 104560821 B CN104560821 B CN 104560821B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
Abstract
The invention provides a kind of Nitrobacter winogradskyi XY 01, preserving number is CGMCC 10171, the Nitrobacter winogradskyi XY 01 can use the method for batch feeding to carry out fermented and cultured, cultured products can be used for the removal of cultivation water nitrite nitrogen, can effectively solve the problems, such as nitrite accumulation present in breeding water body, fishes and shrimps yield is improved, is had wide practical use in aquaculture field.
Description
Technical field
The present invention relates to biological technical field, especially a kind of Nitrobacter winogradskyi (Nitrobacter
Winogradskyi) XY-01 and its cultural method and application.
Background technology
Nitrite is the salt of nitrous acid, contains nitrite ion NO2 -, it is poisonous, there is carcinogenicity, in addition to silver nitrate,
It is general all soluble in water.Alkali metal salt is relatively stable, and other salt all decompose when heating.Not only it is oxidizing but also have reproducibility.It is a large amount of to use
In chemical synthesis, food service industry and the chemical reagent indispensable as laboratory.
In aquaculture, fishes and shrimps residual bait and its excreta decompose in water and generate substantial amounts of ammonia and noxious material, then
Nitrous acid is converted into quickly by the effect of nitrococcus and photosynthetic bacteria, and nitrous acid is combined with the metal ion in water body and can produced
Raw nitrite, in addition, bottom of pond mud is not removed for a long time, cultivation density, which crosses conference, causes water-bed anoxic, in microorganism
Under effect, itrogenous organic substance decomposes, and can be also present in the form of nitrite in water body causes nitrite accumulation.It is aquatic dynamic
Thing absorbs excessive nitrite salt, can influence the running of oxygen carrying cell in blood, it is suppressed that the oxygen carrying capability of blood, can seriously cause
Fishes and shrimps mortality.
It can be seen that nitrite accumulation is to cause the main original that the aquatic animal such as fishes and shrimps is poisoned to death in high-density aquiculture
Therefore one, seek effective nitrite degradation method, control cultivation later stage pond body nitrite concentration be improve cultivation into
Power, increase the only way which must be passed of per mu yield.
Although traditional oxidizing process or reducing process can safely and effectively reduce nitrite concentration, there is during maintenance
Between short, the shortcomings of water nitrite easily rebounds；Although physisorphtion has, action time is short, low cost and other advantages,
Need the dosage of adsorbent huge；By changing the shortcomings that indirect methods such as water are handled obviously there is also curing the symptoms, not the disease.
Therefore, although common physical-chemical process can have certain effect, there is medicament requirement it is big, maintain when
Between it is short, it is easy bounce-back etc. problems, therefore, screening be capable of effectively degrading nitrite bacterial strain and explore its High Density Cultivation side
Method, turn into one of aquaculture field problem urgently to be resolved hurrily.
Nitrobacter winogradskyi disclosed by the invention can efficiently degrade the nitrite in water body under aerobic condition, reduce
Nitrite nitrogen concentration, therefore will be made for the development of micro- pollution of ecological environment biologic treating technique in aquaculture positive
Contribution.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of Nitrobacter winogradskyi XY-01, and can effectively degrade water body
In nitrite.
Another technical problem to be solved by this invention is to provide the culture side for above-mentioned Nitrobacter winogradskyi XY-01
Method.
Another technical problem to be solved by this invention is to provide for preparing containing above-mentioned Nitrobacter winogradskyi XY-01's
The method of microbial inoculum and its microbial inoculum prepared by this method.
Another technical problem to be solved by this invention is to provide above-mentioned Nitrobacter winogradskyi XY-01 or containing above-mentioned Vickers
The application of bacterium nitrobacter XY-01 microbial inoculum.
In order to solve the above technical problems, the technical scheme is that：
The present invention provides a kind of Nitrobacter winogradskyi (Nitrobacter winogradskyi) XY-01, and its preserving number is
(the depositary institutions of CGMCC 10171:China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：Court of Beijing
The positive institute 3 of area's North Star West Road 1, preservation date：On December 15th, 2014, deposit number：CGMCC 10171).
Preferably, above-mentioned Nitrobacter winogradskyi XY-01, by being screened in sludge sewage, its bacterium colony exists
Transparent circular fine particle shape, Gram's staining are negative on culture plate, are nitrified by 16SrDNA sequencing identifications for Vickers
Bacillus, 16SrDNA sequences are as shown in SEQ ID NO.1.
Preferably, above-mentioned Nitrobacter winogradskyi XY-01, there is stronger nitrite degradation ability.
Preferably, above-mentioned Nitrobacter winogradskyi XY-01, concentration range Wei≤615mg/L of degradable nitrite nitrogen, it is excellent
Select ,≤400mg/L, preferred ,≤300mg/L, most preferred ,≤200mg/L.
It is furthermore preferred that nitrite nitrogen degradation rates of the Nitrobacter winogradskyi XY-01 in its nutrient solution can reach
More than 60mg-N/ (Lh).It is preferred that up to 73mg-N/ (Lh)
The present invention also provides a kind of culture medium for above-mentioned Nitrobacter winogradskyi XY-01, and component is as follows：In every 1L water,
Wherein, solion component：Contain FeSO per 1L water4·7H2O 1g, EDTA 1g.
Preferably, the nutrient media components is：
In every 1L water,
Wherein, solion component：Contain FeSO per 1L water4·7H2O 1g, EDTA 1g.
It is furthermore preferred that the nutrient media components is：
In every 1L water,
Wherein, solion component：Contain FeSO per 1L water4·7H2O 1g, EDTA 1g.
Preferably, above-mentioned culture medium, it is prepared by following methods：Above-mentioned each component uses water after being weighed by prescription
Standby culture medium is made in dissolving, and sterilize 20-30min under 121 DEG C, 0.11Mpa.
The present invention also provides a kind of bacterium nitrobacter XY-01 cultural method, is cultivated with above-mentioned culture medium.
Preferably, above-mentioned Nitrobacter winogradskyi XY-01 is cultivated using batch feeding mode.It is furthermore preferred that specific behaviour
It is as method：Using the nitrite nitrogen concentration in spectrophotometry nutrient solution, when nitrite nitrogen concentration is less than 30mg/
During L, sodium nitrite solution to the nutrient solution nitrogen concentration of nitrite for supplementing 40% is 200-400mg/L or so, and culture is extremely
OD600Terminate to cultivate when reaching more than 0.3.
The present invention also provides a kind of purposes of above-mentioned bacterium nitrobacter XY-01 in water body processing microbial inoculum is prepared.
The present invention also provides a kind of microbial inoculum, wherein, the microbial inoculum includes the bacterium nitrobacter XY-01 bacterial strains of above-mentioned separation.It is preferred that
, the microbial inoculum also includes stabilizer and protective agent.
The present invention also provides a kind of preparation method of microbial inoculum, wherein, the microbial inoculum prepared by above-mentioned bacterium nitrobacter XY-01 and
.Preferably, the preparation method includes the concentration of (1) bacterium nitrobacter XY-01 zymotic fluids；(2) stabilizer and protective agent are added.
Preferably, the method for concentration of zymotic fluid is to be concentrated into the zymotic fluid obtained at the end of culture using tube centrifuge
The 1/10 of original volume, its nitrite nitrogen degradation capability is set to reach more than 600mg-N/ (Lh), preferably up to 730mg-N/
(L·h)。
Preferably, stabilizer is final concentration of 0.3 ‰ natrium nitrosum.Protective agent is preferably glycerine, sorbierite, dimethyl
Any of sulfoxide, trehalose are a variety of.
The present invention also provides a kind of application of above-mentioned bacterium nitrobacter XY-01 bacterial strains or the microbial inoculum in water body processing.
The present invention also provides a kind of water body processing method, is included in water body and launches above-mentioned XY-01 bacterial strains or above-mentioned bacterium
Agent.Preferably, under aerobic condition, the effect of its degrading nitrite is realized.
Preferably, the water body is breeding water body, it is furthermore preferred that the breeding water body is the cultivation water rich in nitrite
Body.Preferably, concentration range Wei≤615mg/L of Nitrite nitrogen, You select ,≤400mg/L, and preferred ,≤
300mg/L, most preferred ,≤200mg/L.
Preferably, the inoculum concentration of the bacterial strain or microbial inoculum is that (inoculative proportion is per cubic meter of water 6-2500mL
0.006 ‰ -2.5 ‰), and preferable inoculum concentration is per cubic meter of water 10-2500mL (inoculative proportion is 0.01 ‰ -2.5 ‰), more
Preferable inoculum concentration is per cubic meter of water 100-2500mL (inoculative proportion is 0.1 ‰ -2.5 ‰), wherein bacterial strain or microbial inoculum
OD600At least 3, preferably greater than 3, its nitrite nitrogen degradation capability reaches more than 600mg-N/ (Lh), preferably reachable
730mg-N/(L·h)。
The beneficial effects of the invention are as follows：
The invention provides a kind of Nitrobacter winogradskyi XY-01 (preserving number is CGMCC 10171), and it can realize that it is high
Density culture, the degraded of breeding water body nitrite being can be used for after cultured products are concentrated, the nitrite concentration of degraded is high,
Degradation rate is fast, and clearance is close to 100%.Compared with physics, chemical method have effect persistently, do not introduce other pollutants etc.
Advantage, have wide practical use in aquaculture field.
Preservation information
Classification noun：Nitrobacter winogradskyi (Nitrobacter winogradskyi)
Depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On December 15th, 2014
Preserving number：CGMCC 10171
Brief description of the drawings
Fig. 1 is Nitrobacter winogradskyi XY-01 nutrient solution nitrite nitrogen degradation curves
Fig. 2 is nitrite nitrogen degradation curve under the conditions of Nitrobacter winogradskyi XY-01 inoculations compare 2.5 ‰
Fig. 3 is nitrite nitrogen degradation curve under the conditions of Nitrobacter winogradskyi XY-01 inoculations compare 0.01 ‰
Embodiment
Technical scheme of the present invention is further described with reference to specific embodiment.
Strain used is Nitrobacter winogradskyi XY-01 in the following embodiments, different culture medium in embodiment
And solution formula is as follows：
1. enriched medium：NaNO20.5g, NaCl 0.3g, MgSO40.14g, KH2PO40.136g, NaHCO31.6g
FeSO40.03g, distilled water 1000mL, pH 8.4
2. fermentation medium 1：Na2HPO41.7g, KH2PO40.18g, NaNO20.3g, MgSO4·7H2O 0.01g,
CaCL20.001g, solion 1mL, pH 7.3,
Solion component：FeSO4·7H2O 1g, EDTA 1g, water 1L.
3. fermentation medium 2：Na2HPO42.55g KH2PO40.27g, NaNO21.2g, MgSO4·7H2O 0.02g,
CaCL20.0019g, solion 10mL, pH 8.4,
Solion component：FeSO4·7H2O 1g, EDTA 1g, water 1L.
4. fermentation medium 3：Na2HPO43.2g, KH2PO40.36g, NaNO23g, MgSO4·7H2O0.03g,
CaCL20.003g, solion 20mL, pH 9,
Solion component：FeSO4·7H2O 1g, EDTA 1g, water 1L.
5. beef extract-protein culture medium：Beef extract-protein culture medium：Beef extract 3g/L；Peptone 10g/L；Chlorination
Sodium 5g/L, pH 7.4~7.6.
6.LB culture mediums：Tryptone 10g/L；Yeast extract 5g/L；Sodium chloride 5g/L, pH 7.0.
7. micro-polluted water produces cultivation water：It is taken from Baodi District, Tianjin City tropical fish culture pond and Hangu District Penaeus Vannmei is supported
Grow pond.
The Nitrobacter winogradskyi XY-01 of embodiment 1 screening and identification
Aquaculture pond bed mud sample 1g is taken to be dissolved in the 100mL concentration and separation culture mediums prepared, in 30 DEG C of insulating boxs
Culture to inspection does not measure nitrite nitrogen under conditions of middle 180r/min, then draws 5mL cultured products and is transferred to new culture
Continue to cultivate in base, 3 times repeatedly, cultured products are finally pressed into gradient dilution, 10-1、10-2... until being diluted to 10-7, Mei Genong
Degree is lower to apply flat board, and picking single bacterium colony repeats line purifying 2-3 times, obtains purifying on bacterial strain access inclined-plane, through beef extract-peptone
Culture medium and the checking of LB culture mediums grow without heterotroph；Its bacterium colony transparent circular fine particle shape on culture plate, leather are blue
Albert'stain Albert is negative, and by 16SrDNA sequencing identifications, is defined as Nitrobacter winogradskyi, and the sequence of bacterium 16SrDNA sequencings is such as
Under：
SEQ ID NO.1：
GTCGGCTGCCTCCCTTGCGGGTTAGCGCACCGCCTTCAGGTAAAACCAACTCCCATGGTGTGACGGGCGGTGTGTAC
AAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGGGCTCGGGTTGCA
GAGCCCAATCCGAACTGAGACGGTTTTTTGAGATTTGCTAGGGGTCACCCCTTTGCTTCCCATTGTCACCGCCATTG
TAGCACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCGCGGCTTATCACCGG
CAGTCTCCTTAGAGTGCTCAACTAAATGGTAGCAACTAAGGACGGGGGTTGCGCTCGTTGCGGGACTTAACCCAACA
TCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGCTCCATGCTCCGAAGAGAAGGTCACATCTCTGCGACC
GGTCATGGACATGTCAAGGGCTGGTAAGGTTCTGCGCGTTGCGTCGAATTAAACCACATGCTCCACCGCTTGTGCGG
GCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGCTTAAAGCGTTAGCTGCGCCA
CTAGTGAGTAAACCCACTAACGGCTGGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTC
CCCACGCTTTCGTGCCTCAGCGTCAGTATCGGGCCAGTGAGCCGCCTTCGCCACTGGTGTTCTTGCGAATATCTACG
AATTTCACCTCTACACTCGCAGTTCCACTCACCTCTCCCGAACTCAAGATCCTCAGTATCAAAGGCAGTTCTGGAGT
TGAGCTCCAGGATTTCACCCCTGACTTAAAGACCCGCCTACGCACCCTTTACGCCCAGTAATTCCGAGCAACGCTAG
CCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTATTCTTGCGGTACCGTCATTATCTTCCCGC
ACAAAAGAGCTTTACAACCCTAGGGCCTTCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAAT
ATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCTGATCATCCTCTCAGACCAGC
TACTGATCGTCGCCTTGGTGAGCCGTTACCTCACCAACAAGCTAATCAGACGCGGGCCGATCTTTCGGCGATAAATC
TTTCCCCGTAAGGGCTTATCCGGTATTAGCCCAAGTTTCCCTGGGTTGTTCCGAACCAAAAGGTACGTTCCCACGCG
TTACTCACCCGTCTGCCACTGACGTATTGCTACGCCCGTTCGACTGCATGGTA。
The Nitrobacter winogradskyi XY-01 degrading nitrites of embodiment 2
Specific implementation step is as follows：Inoculation is into fermentation medium 1,30 DEG C of shaken cultivations, according to《Water quality nitrous acid
The measure AAS (GB7493-87) of salt nitrogen》The nitrite nitrogen concentration in nutrient solution is determined, when nitrite nitrogen concentration
During less than 30mg/L, sodium nitrite solution to the nutrient solution nitrogen concentration of nitrite for supplementing 40% is 200-400mg/L or so,
Dense to bacterium is OD600Reach more than 0.3 end culture, 9000r/min is collected by centrifugation thalline, is all forwarded to same amount of fermentation
In culture medium 2 (nitrite nitrogen concentration is about 240mg/L), 30 DEG C, 150r/min shaking table shaken cultivations, 5mL is sampled every 1h,
9000r/min centrifuging and taking supernatants, determine nitrite nitrogen concentration, draw nitrite nitrogen degradation curve, as a result as shown in figure 1,
The nitrite nitrogen degradation rate of nutrient solution is 73mg-N/ (Lh) as seen from the figure.
The Nitrobacter winogradskyi XY-01 degrading nitrites of embodiment 3
Specific implementation step is as follows：Inoculation is into fermentation medium 2,30 DEG C of shaken cultivations, according to《Water quality nitrous acid
The measure AAS (GB7493-87) of salt nitrogen》The nitrite nitrogen concentration in nutrient solution is determined, when nitrite nitrogen concentration
During less than 30mg/L, sodium nitrite solution to the nutrient solution nitrogen concentration of nitrite for supplementing 40% is 200-400mg/L or so,
Dense to bacterium is OD600Reach more than 0.3 end culture, it is OD to be forwarded to fermentation tank it is dense to bacterium to expand culture600Reach more than 0.3,
After tube centrifuge concentration, bacterium is dense to increase to OD600≈ 3.0, fermented with 2.5 ‰ inoculation 0.5mL bacterium solutions to 200mL
In culture medium 3,30 DEG C, 150r/min shaking table shaken cultivations, 5mL is sampled daily, 9000r/min centrifuging and taking supernatants, determines nitrous
Hydrochlorate nitrogen concentration, nitrite nitrogen degradation curve is drawn, as a result as shown in Fig. 2 as seen from the figure, inoculation is than the condition for 2.5 ‰
Under, strain X Y-01 is seeded to nitrite nitrogen concentration as that after 613mg/L culture mediums, can rapidly adapt to environment, can be by Asia in 3 days
Nitrate nitrogen concentration is down to 0.2mg/L, and clearance is close to 100%.
The Nitrobacter winogradskyi XY-01 degrading nitrites of embodiment 4
Deal with objects to be taken from the water body of Baodi District, Tianjin City tropical fish culture pond, the method for operation is to be aerated in 2L measuring cups
Culture.
Specific implementation step is as follows：For inoculation into fermentation medium 1, it is OD that 30 DEG C of shaken cultivations are dense to bacterium600Reach
More than 0.3, it is OD to be forwarded to fermentation tank it is dense to bacterium to expand culture600Reach more than 0.3, after tube centrifuge concentration, bacterium
It is dense to increase to OD600≈ 3.0, the 0.2mL bacterium solutions are inoculated with equipped with 2L Baodi Districts tropical fish culture pond water with 0.1 ‰ inoculum concentrations
In measuring cup, while blank control is set, and set one group it is parallel, totally 4 groups, aeration operation 3 days under room temperature (20-25 DEG C)；Respectively at
React 24h, 48h and 72h is sampled, 9000r/min centrifuging and taking supernatants, according to《The measure light splitting of water quality nitrite nitrogen
Photometry (GB7493-87)》Determine nitrite nitrogen.As a result as shown in table 1, as shown in Table 1, inoculation is than the condition for 0.1 ‰
Under, the nitrite in strain X Y-01 breeding water bodies capable of being fast degraded, nitrite nitrogen concentration 0.1mg/L can be down in 3 days
Left and right, effectively eliminate influence of the nitrite accumulation to aquatile.
The Nitrobacter winogradskyi XY-01 lab scale nitrite nitrogen degradation datas of table 1.
The Nitrobacter winogradskyi XY-01 degrading nitrites of embodiment 5
Deal with objects to be taken from the water body on the Tianjin Hangu District culture of Penaeus vannamei pool, the method for operation is 20L measuring cups
Interior aeration culture.
Specific implementation step is as follows：For inoculation into fermentation medium 2, it is OD that 30 DEG C of shaken cultivations are dense to bacterium600Reach
More than 0.3, it is OD to be forwarded to fermentation tank it is dense to bacterium to expand culture600Reach more than 0.3, after tube centrifuge concentration, bacterium
It is dense to increase to OD600≈ 3.0, the 0.2mL bacterium solutions are inoculated with respectively at equipped with 20L Hangu District Penaeus Vannmeis with 0.01 ‰ inoculum concentrations
Culture pond water and fermentation medium 1 (nitrite nitrogen concentration be adjusted to the breeding water body similar in 10mg/L or so) measuring cup
In, while blank control is set, and totally 3 groups, aeration operation 14 days under outdoor (15-20 DEG C)；Daily sampling 5mL, 9000r/min from
The heart takes supernatant, according to《The measure AAS (GB7493-87) of water quality nitrite nitrogen》Determine nitrite nitrogen.As a result
As shown in figure 3, as seen from the figure, inoculation than under conditions of for 0.01 ‰, 15-20 DEG C of temperature, strain X Y-01 be seeded to culture medium and
After breeding water body, all in the presence of certain lag phase, in the medium, lag phase is 4 days, in breeding water body, lag phase 8
My god, but after lag phase, the nitrite nitrogen in system quickly can be down to 0.1mg/L or so.
It is prepared by the Nitrobacter winogradskyi XY-01 microbial inoculums of embodiment 6
Specific implementation step is as follows：Inoculation is into fermentation medium 1,30 DEG C of shaken cultivations, according to《Water quality nitrous acid
The measure AAS (GB7493-87) of salt nitrogen》The nitrite nitrogen concentration in nutrient solution is determined, when nitrite nitrogen concentration
During less than 30mg/L, sodium nitrite solution to the nutrient solution nitrogen concentration of nitrite for supplementing 40% is 200-400mg/L or so,
Dense to bacterium is OD600Reach more than 0.3 end culture, be concentrated into the 1/10 of original volume through tube centrifuge, bacterium is dense to increase to OD600
≈ 3.0, nitrite nitrogen degradation capability reach more than 600mg-N/ (Lh), add 0.3 ‰ natrium nitrosum as stabilizer,
Add glycerine, sorbierite, dimethyl sulfoxide (DMSO), any of trehalose or it is a variety of be used as protective agent, that is, obtained Vickers nitrification
Bacillus XY-01 microbial inoculums.
The Nitrobacter winogradskyi XY-01 microbial inoculum degrading nitrites of embodiment 7
It is 9.8mg/L to deal with objects as the cultivation water on Hangu District South America, the Tianjin white shrimp pool, its nitrite nitrogen.Operation side
Formula is 4500m3Field adds experiment, and surface aeration machine is set in the middle part of pond body and carries out Air Exposure.Experimental stage is the cultivation later stage, right
Shrimp floating head phenomenon is frequent, using chemical enhancement agent prawn need to be prevented because being choked to death daily.
Specific implementation step is as follows：27L Nitrobacter winogradskyi XY-01 microbial inoculums are inoculated with 4500m with 0.006 ‰ inoculum concentrations3
In leg Shrimp Litopenaeus vannamei, with aerator among crop field Air Exposure；Respectively at 2d, 5d, 10d,
15d, 25d are sampled, 9000r/min centrifuging and taking supernatants, according to《Measure AAS (the GB7493- of water quality nitrite nitrogen
87)》Determine nitrite nitrogen.As a result as shown in table 2, as seen from table, the nitrite nitrogen concentration after microbial inoculum in pond body is added to hold
It is continuous to reduce, from 5d, chemical enhancement agent is stopped using, until 25d cultivation end is prawn floating head phenomenon occur.
The Nitrobacter winogradskyi XY-01 field experiment nitrite nitrogen degradation datas of table 2.
| Number of days | Nitrite nitrogen (mg/L) | Remarks |
| 0 | 9.8 | Add Nitrobacter winogradskyi XY-01 microbial inoculums and amount to 27L |
| 2 | 7.5 | |
| 5 | 5.1 | |
| 10 | 4.97 | |
| 15 | 4.85 | |
| 25 | 3.62 |
It is above-mentioned that this plant of Nitrobacter winogradskyi XY-01 and its cultural method are retouched in detail with what application was carried out with reference to embodiment
State, be illustrative rather than limited, several embodiments can be included according to limited scope, therefore do not departing from this
Changing and modifications under invention general plotting, it should belong within protection scope of the present invention.
Claims (9)
1. a kind of Nitrobacter winogradskyi (Nitrobacter winogradskyi) XY-01, it is characterised in that：Its preserving number is
CGMCC No.10171。
2. Nitrobacter winogradskyi according to claim 1 (Nitrobacter winogradskyi) XY-01, its feature exists
In：By being screened in sludge sewage, its bacterium colony transparent circular fine particle shape on culture plate, leather orchid
Albert'stain Albert is negative, and is Nitrobacter winogradskyi by 16SrDNA sequencing identifications, 16SrDNA sequences are as shown in SEQ ID NO.1.
3. a kind of any Nitrobacter winogradskyi of claim 1-2 (Nitrobacter winogradskyi) XY-01 training
The method of supporting, it is characterised in that：It is cultivated using following culture medium：
Culture medium prescription is as follows：In every 1L water,
Solid-state adds 1.5% agar,
Solion component：Contain FeSO per 1L water4·7H2O 1g, EDTA 1g, water 1L.
4. the cultural method described in claim 3, it is characterised in that：Cultivated using batch feeding mode, concrete operations side
Method is：Using the nitrite nitrogen concentration in spectrophotometry nutrient solution, when nitrite nitrogen concentration is less than 30mg/L,
Sodium nitrite solution to the nutrient solution nitrogen concentration of nitrite of supplement 40% is 200-400mg/L, is cultivated to OD600Reach 0.3
Terminate to cultivate during the above.
5. any described Nitrobacter winogradskyis of claim 1-2 (Nitrobacter winogradskyi) prepared by XY-01
Purposes in water body processing microbial inoculum.
A kind of 6. microbial inoculum, it is characterised in that including any described Nitrobacter winogradskyis of claim 1-2 (Nitrobacter winogradskyi)XY-01。
7. a kind of preparation method of microbial inoculum, the preparation method includes the Vickers that (1) preserving number is CGMCC No.10171 and nitrifies bar
Bacterium (Nitrobacter winogradskyi) XY-01 zymotic fluids concentration；(2) stabilizer and protective agent are added.
8. any described Nitrobacter winogradskyis of claim 1-2 (Nitrobacter winogradskyi) XY-01 or right
It is required that the microbial inoculum described in 6 is in the application of water body Nitrite Nitrogen of degrading.
9. a kind of water body processing method, it is characterised in that be included in water body and launch any described Vickers nitre of claim 1-2
Change bacillus (Nitrobacter winogradskyi) XY-01 or the microbial inoculum described in claim 6.
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| CN105543093B (en) * | 2015-12-22 | 2019-10-01 | 天津凯英科技发展股份有限公司 | A kind of ammonia nitrogen degradation microbial inoculum preservative agent and store method |
| CN108641975A (en) * | 2018-04-13 | 2018-10-12 | 北京天诚众合科技发展有限公司 | A kind of breeding water body water purification agent and preparation method thereof |
| CN110465184A (en) * | 2019-08-30 | 2019-11-19 | 天津凯英科技发展股份有限公司 | Deodorant filler and preparation method thereof, deodorization device and deodorizing methods and application |
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| 低温硝化细菌的筛选及特性研究;陈中祥等;《江苏农业科学》;20111231;284 -286 * |
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