CN107858311A - A kind of Achromobacter xylosoxidans X10 and its application - Google Patents
A kind of Achromobacter xylosoxidans X10 and its application Download PDFInfo
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- CN107858311A CN107858311A CN201711286064.2A CN201711286064A CN107858311A CN 107858311 A CN107858311 A CN 107858311A CN 201711286064 A CN201711286064 A CN 201711286064A CN 107858311 A CN107858311 A CN 107858311A
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- C—CHEMISTRY; METALLURGY
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- 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
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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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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/00—Nature of the contaminant
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- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The invention belongs to biology techniques field, a kind of Achromobacter xylosoxidans X10 and its application are disclosed, the Achromobacter xylosoxidans X10 of degrading nitrite, preservation is entitled：Achromobacter xylosoxidans X10, depositary institution：China typical culture collection center, preservation address：Wuhan, China, Wuhan University；Preservation date：On September 22nd, 2017, preserving number：CCTCC M2017535；The application of nitrite nitrogen contents of the Achromobacter xylosoxidans X10 of the effectively degrading nitrite in breeding water body is reduced.The Achromobacter xylosoxidans culture of the effectively degrading nitrite of the present invention is convenient, and the speed of growth is fast；PH and temperature tolerance range are wide；Viable bacteria number of strains after cultivation and fermentation is high；The bacterium can effectively remove the nitrite nitrogen in breeding water body, significantly improve water quality.
Description
Technical field
The invention belongs to biology techniques field, more particularly to a kind of Achromobacter xylosoxidans X10 and its application.
Background technology
Culture fishery development is swift and violent, and scale is increasing, and breed variety, pattern are varied.Intensive culture pattern
Under, fish has been fed overfeeding, is deposited on bottom, and efficiency of feed utilization is low in addition, and the excreta of cultivation product itself influences, past
It is past to make breeding water body Central Asia nitrate nitrogen content severe overweight.NO2 -Protoferriheme is combined into ferroheme in animal body, it is high
Ferri-hemoglobin can not deliver oxygen, cause fish hypoxia death.Meanwhile water body Central Asia nitrate nitrogen content has with explosive fish disease
Substantial connection.Under normal circumstances, the nitrite nitrogen in breeding water body should be controlled in below 0.2mg/L, the meeting in 0.5mg/L
Cause death.Therefore the biology of aquaculture system nitrite nitrogen eliminates prominent urgently as energy one in aquatic product health cultivation
The problem of to be solved.
Degrading nitrite product in current cultivation is all product (such as polymerization of absorbent-type and ion-exchanger type
The products such as aluminium chloride, alum, sodium thiosulfate, cpolyacrylamidses), these materials are containing can be dense by the nitrite in water body
Polycondensation combines in adsorbent and falls to bottom of pond, the result is that water middle level and top layer nitrite concentration reduce, but the Asia of bottom
Nitrate is significantly raised, and its harm is serious all the better：First, endangering benthon existence, second, when meeting the change of weather, easily occur
" returning bottom ", trigger massive mortality.Bioremediation technology based on microbial degradation is to eliminate environment nitrite
Effective way, research and application about nitrite degradation bacterial strain are more, and nitrite degradation bacterium mainly has Nitrobacter
(Nitrobacter sp.), Nitraspira (Nitrospira sp.) and Nitrococcus (Nitrococcus sp.), vacation
Zygosaccharomyces (Pseudomonas sp.), photosynthetic bacteria (Rhodopseudomonas sp.) etc..In China, American-European, day
In the culture fishery of the country such as this and Indonesia, Thailand, probiotics obtains relatively broad application.But
Probiotics viable bacteria concentration in production practices can not usually keep stable, easily be influenceed by outside environmental elements.Such as water body
Temperature, pH, dissolved oxygen etc. influence.
In summary, the problem of prior art is present be：
Presently used probiotics has that beneficial bacterium strain species is on the low side, living bacteria count is low, tolerance is not strong etc. and lacked
Fall into, it is impossible to meet the needs in practice, constrain application of the probiotics on aquaculture water purifying.
The content of the invention
The problem of existing for prior art, the invention provides a kind of Achromobacter xylosoxidans X10 and its application.
The present invention is achieved in that, the invention provides a kind of Achromobacter xylosoxidans, its preservation is entitled：
Achromobacter xylosoxidans X10, depositary institution：China typical culture collection center, preservation address：China
Wuhan, Wuhan University；Preservation date：On September 22nd, 2017, preserving number：CCTCC M2017535.
Present invention also offers the purposes of above-mentioned Achromobacter xylosoxidans：For purifying aquaculture waste water.
Bacterial strain (A.xylosoxidans) X10 of the present invention is in uniform muddiness in nitrite screens fluid nutrient medium,
Colony diameter is 2.5-3.0mm on nitrite screening agar medium flat board, circular, ecru, and surface is smooth impermeable
Bright, glossy, centre slightly projection, neat in edge, no halo, quality are softer.It is in uniform muddiness in fluid nutrient medium is nitrified,
Stationary phase bacterial strain concentration may be up to 9 × 108cfu/mL。
Achromobacter xylosoxidans (A.xylosoxidans) X10 of the present invention is only nitrogen source in nitrite
In (500mg/L) screening and culturing medium, when 30 DEG C, under the conditions of 180rpm/min, 48h accumulations reduce nitrite and reach 310mg/L；
In its most suitable nitrite concentration 300mg/L, degradation rate 100%；Low concentration nitrite (5.0,2.0,1.0,0.5,
0.2nd, 0.1,0.05,0.025mg/L) under the conditions of, after being inoculated with 24h, nitrite clearance is up to more than 99%.
Advantages of the present invention and good effect are：
The Achromobacter xylosoxidans culture of the effectively degrading nitrite of the present invention is convenient, and the speed of growth is fast；
Viable bacteria number of strains after cultivation and fermentation is high；
Stronger nitrogen removal performance is still kept in pH value 5.0-9.0；
Stronger nitrogen removal performance is still kept in 25-45 DEG C of temperature；
The bacterium can effectively remove the nitrite nitrogen in breeding water body, significantly improve water quality.
During the A.xylosoxidans X10 actual uses of the present invention, the method launched into water, general control can be taken
Concentration of the A.xylosoxidans X10 in water body is 1 × 107cfu/mL。
Brief description of the drawings
Fig. 1 be Achromobacter xylosoxidans provided in an embodiment of the present invention (A.xylosoxidans) X10 growth curves with
Nitrite clearance.
Fig. 2 is Achromobacter xylosoxidans provided in an embodiment of the present invention (A.xylosoxidans) X10 sub- in high concentration
48h clearances under the conditions of nitrate.
Fig. 3 is Achromobacter xylosoxidans provided in an embodiment of the present invention (A.xylosoxidans) X10 sub- in low concentration
24h clearances under the conditions of nitrate.
Fig. 4 is Achromobacter xylosoxidans provided in an embodiment of the present invention (A.xylosoxidans) X10 in different pH bars
48h clearances under part.
Fig. 5 is Achromobacter xylosoxidans provided in an embodiment of the present invention (A.xylosoxidans) X10 different temperatures bars
48h clearances under part.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to this hair
It is bright to be further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Below in conjunction with the accompanying drawings and specific embodiment is described in detail to the application principle of the present invention.
Embodiment 1,
Achromobacter xylosoxidans (A.xylosoxidans) X10 provided in an embodiment of the present invention acquisition, is carried out successively
Following steps：
(1), the screening of bacterium, isolate and purify：
In Poyang Lake and letter river intersection (N28 ° 49 ' 46.69 " east longitudes of north latitude
E116 ° 22 ' 32.77 "), fetch water 500ml and bed mud 300g.After fully mixing, nitrobacteria richness is inoculated into by 5%
Collect culture medium (Na NO21.0g, CH3COONa 4.8g, K2HPO4.3H2O 1.2g, Fe3PO4.4H2O 0.01g) in 180rpm, 30
After DEG C culture 48h, then nutrient solution is inoculated into nitrobacteria enriched medium by 5%, after 48h, takes 100 μ L bacterium solutions to be coated with
In nitrobacteria enrichment agar medium, after 30 DEG C are incubated 48h, monoclonal is selected, is drawn in nitrobacteria enrichment agar medium
Line, after 30 DEG C are incubated 48h, 240 plants of monoclonal is selected, is inoculated into nitrobacteria screening and culturing medium (NaNO20.5g, CH3COONa
3.4g, K2HPO4. 3H2O 1.0g, Fe3PO4.4H2O 0.01g, Mg SO4.7H2O 0.01g, NaCl 30g), 180rpm, 30 DEG C
After cultivating 48h, 63 plants of the good bacterial strain of growing state is filtered out, is mixed with 40% sterile glycerol (mass ratio) isometric (500 μ L)
Uniformly preserved in -80 DEG C of refrigerators.
(2), the nitrite degradation effect test of bacterium：
The bacterial strain of separation in step (1) is activated in LB culture mediums (peptone 10g, NaCl 10g, yeast extract 5g)
Afterwards, nitrobacteria screening and culturing medium is inoculated into, 180rpm, 30 DEG C of cultures, 2ml bacterium solutions, 12000rpm centrifugations are taken in 24h, 48h
10min, supernatant is taken, blank nitrobacteria screening and culturing medium is control, utilizes kit measurement supernatant NO2 -Concentration, calculate 63 plants
Bacterium nitrite degradation rate.As a result show, the degradation efficiency highest of strain X 10, up to 62%.X10 is bacterial strain of the present invention.
(3), strain idenfication：
1), form is extraordinary
In uniformly muddiness in nitrite screens fluid nutrient medium, on nitrite screening agar medium flat board
Colony diameter is 2.5-3.0mm, and circular, ecru, surface is smooth opaque, glossy, centre slightly projection, neat in edge, nothing
Halo, quality are softer.
2), 16S rDNA sequence analyses：
16S rDNA identifications are carried out to the X10 being separated to.Primer sequence is general 16S rDNA primer 2s 7f (SEQ ID
NO.1：5'-AGAGTTTGATCCTGGCTCAG -3') and 1492r (SEQ ID NO.2：
5'—GGTTACCTTGTTACGACTT—3').Synthesized by raw work bioengineering Shanghai Co., Ltd.To screen
To the STb gene of bacterial strain enter performing PCR amplification for template, amplification reaction system (50 μ L) is：The μ L, dNTP 4.0 of 10 × buffer 5
μ L, upstream and downstream primer each μ L of 1.0 μ L, taq enzyme 0.5, template 1.0 μ L, ddH2O 37.5μL.Response procedures：95℃5min；94℃
30s, 54 DEG C of 30s, 72 DEG C of 30s, 32 circulations；72 DEG C of extension 10min.Purpose fragment send Hua Da gene sequencing, sequencing result with
16S rDNA sequences (MF144503.1, MF144475.1, KX350007.1, the KX349993.1 logged in GeneBank
Deng) be compared, homology reaches 99%.By morphological feature and 16S rDNA sequence analyses, determine that strain X 10 is
Achromobacter xylosoxidans (A.xylosoxidans).
Carried out preservation, the entitled Achromobacter xylosoxidans X10 of preservation, depositary institution：China
Type Tissue Collection, preservation address：Wuhan, China, Wuhan University；Preservation date：On September 22nd, 2017, preserving number：
CCTCC M2017535。
Embodiment 2
Achromobacter xylosoxidans (A.xylosoxidans) X10 nitrogen removal characteristics provided in an embodiment of the present invention, enter successively
Row following steps：
In the final concentration of 500mg/L of nitrite heterotrophic nitrification culture medium, trained with 3% inoculum concentration access in LB
The X10 seed liquors (A600nm ≈ 0.4) cultivated in base are supported, in 180r/min, 30 DEG C, constant-temperature table concussion and cultivate.Taken every 3h
Sample, determine the increment and nitrite clearance of thalline.
Concrete outcome is shown in accompanying drawing 1：In logarithmic phase, X10 nitrite clearance rises and raised with concentration, laggard in 42h
Enter stationary phase, nitrite clearance reaches highest 63%, then begins to slowly decline.
Different content natrium nitrosum added in heterotrophic nitrification culture medium make its concentration be respectively 25,50,75,100,
200、300、400、500、600、700mg/L.The X10 seed liquors cultivated with 3% inoculum concentration access in LB culture mediums
(A600nm ≈ 0.4), in 180r/min, 30 DEG C, constant-temperature table concussion and cultivate.Sampled after 48h, determine thalline increment and
Nitrite clearance.Concrete outcome is shown in accompanying drawing 2：In different nitrite culture mediums, X10 growth differences unobvious, in Asia
When nitrate is less than 300mg/L, nitrite clearance is 100%.
Culture medium nitrite is adjusted to 5.0 close to aquaculture wastewater, 2.5,1.0,0.5,0.2,0.1,
0.05、0.025mg/L.The X10 seed liquors (A600nm ≈ 0.4) cultivated with 3% inoculum concentration access in LB culture mediums, in
180r/min, 30 DEG C, constant-temperature table concussion and cultivate.Sampled after 24h, determine the concentration of nitrite.Concrete outcome is shown in accompanying drawing 3：
Each concentration cultures nitrite clearance is up to more than 99%.
Embodiment 3,
Achromobacter xylosoxidans (A.xylosoxidans) X10 is to Asia under offer condition of different pH of the embodiment of the present invention
Nitrate removal rate influences, and follows the steps below successively：
In the final concentration of 300mg/L of nitrite, pH is respectively 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,
In 11.0 heterotrophic nitrification culture medium, X10 seed liquors (the A600nm ≈ cultivated in LB culture mediums are accessed with 3% inoculum concentration
0.4), in 180r/min, 30 DEG C, constant-temperature table concussion and cultivate.Sampled after 48h, determine the concentration of nitrite.Concrete outcome
See accompanying drawing 4：X10 denitrogenations Optimal pH is 7.0, and higher nitrite nitrogen removal efficiency is still kept in pH value 5.0-9.0.
Embodiment 4,
The embodiment of the present invention provides X10 pairs of Achromobacter xylosoxidans (A.xylosoxidans) under condition of different temperatures
Nitrite clearance influences, and follows the steps below successively：
In the final concentration of 300mg/L of nitrite, pH 7.0 heterotrophic nitrification culture medium, accessed with 3% inoculum concentration
The X10 seed liquors (A600nm ≈ 0.4) cultivated in LB culture mediums, respectively at 180r/min, 20,25,30,35,40,45 DEG C
Under the conditions of constant-temperature table concussion and cultivate.Sampled after 48h, determine the concentration of nitrite.Concrete outcome is shown in accompanying drawing 5：X10 denitrogenations
Optimal 30-35 DEG C, higher nitrite nitrogen removal efficiency is still kept in 25-45 DEG C of temperature.
Embodiment 5,
Achromobacter xylosoxidans (A.xylosoxidans) X10 provided in an embodiment of the present invention purifies to breeding water body
Effect, is followed the steps below successively：
In LB fluid nutrient mediums, the X10 seed liquors (A600nm cultivated in LB culture mediums is accessed with 3% inoculum concentration
≈ 0.4), in 180r/min, 30 DEG C, constant-temperature table concussion and cultivate 24h.3500r centrifuges 10min, removes culture medium, isometric PBS
Cell is resuspended.Jiangxi Academy of Agricultural Sciences fishing breeding pond water 300L nearby is taken, nitrite concentration is 0.45mg/L after testing.
Waste water is dispensed into 12 buckets, each water bottled water 20L, is randomly divided into 2 groups, 0%, 1%X10 is added respectively and bacterium is resuspended
Liquid.Stood under room temperature (26,30 DEG C), 24,48,72, each bucket water sampling 2mL after 96h, detect NO2 -Concentration.Table specific as follows：
The Achromobacter xylosoxidans of table 1 (A.xylosoxidans) X10 is to cultivation water nitrite removal effect
Embodiment 6
Achromobacter xylosoxidans (A.xylosoxidans) X10 safety evaluatios provided in an embodiment of the present invention：
In LB fluid nutrient mediums, the X10 seed liquors (A600nm cultivated in LB culture mediums is accessed with 3% inoculum concentration
≈ 0.4), in 180r/min, 30 DEG C, constant-temperature table concussion and cultivate 24h.3500r centrifuges 10min, removes culture medium, isometric PBS
Cell is resuspended.X10 cells are added in zebra fish culturing jar, final concentration is about 1 × 107、2×107、3×107、5×
107cfu/mL.7d is observed, it is found that no zebra fish is dead.
Above-mentioned experimental result：Achromobacter xylosoxidans (Achromobacter xylosoxidan are added in breeding water body
S) X10 can significantly reduce nitrite nitrogen content.Show, Achromobacter xylosoxidans (Achromobacterxylosoxidan
S) X10 has the obvious effect for reducing breeding water body nitrite, and temperature and pH tolerance ranges are wide, and to aquatic products
Animal is safe from harm, the probiotics being adapted as in aquaculture.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
<110>Animal and veterinary research institute of Jiangxi Academy of Agricultural Sciences
<120>A kind of Achromobacter xylosoxidans X10 and its application
<160>2
<210>1
<211>20
<212>DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221>CDS
<400>1
AGAGTTTGATCCTGGCTCAG
<210>2
<211>19
<212>DNA
<213>Artificial sequence (Artificial Sequence)
<400>2
GGTTACCTTGTTACGACTT
Claims (7)
1. a kind of Achromobacter xylosoxidans X10, it is characterised in that the Achromobacter xylosoxidans X10, preservation are entitled：
Achromobacter xylosoxidans X10, depositary institution：China typical culture collection center, preservation address：China
Wuhan, Wuhan University；Preservation date：On September 22nd, 2017, preserving number：CCTCC M2017535.
A kind of 2. Achromobacter xylosoxidans X10 degrading nitrite nitrogen contents in breeding water body as claimed in claim 1
Application.
3. Achromobacter xylosoxidans X10 as claimed in claim 2 reduces the application of nitrite nitrogen content in breeding water body,
Characterized in that, the nitrogenous concentration of the Achromobacter xylosoxidans X10 degrading nitrites is less than 300mg/L.
4. Achromobacter xylosoxidans X10 as claimed in claim 2 reduces the application of nitrite nitrogen content in breeding water body,
Characterized in that, the nitrogenous concentration of the Achromobacter xylosoxidans X10 degrading nitrites is：5.0mg/L、2.0mg/L、
1.0mg/L。
5. Achromobacter xylosoxidans X10 as claimed in claim 2 reduces the application of nitrite nitrogen content in breeding water body,
Characterized in that, the nitrogenous concentration of the Achromobacter xylosoxidans X10 degrading nitrites is：0.5mg/L、0.2mg/L、
0.1mg/L。
6. Achromobacter xylosoxidans X10 as claimed in claim 2 reduces the application of nitrite nitrogen content in breeding water body,
Characterized in that, the nitrogenous concentration of the Achromobacter xylosoxidans X10 degrading nitrites is：0.05mg/L、0.025mg/
L。
A kind of 7. Tiny ecosystem suitable for aquaculture prepared using Achromobacter xylosoxidans X10 described in claim 1
Preparation.
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