CN109652568A - A method of fast enriching Nitrosocosmicus belongs to ammoxidation archaeal from environment - Google Patents
A method of fast enriching Nitrosocosmicus belongs to ammoxidation archaeal from environment Download PDFInfo
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Abstract
A kind of method that the present invention discloses the category of the fast enriching Nitrosocosmicus from environment ammoxidation archaeal.This method includes two steps of orienting enriching and fast purifying.Attaching substratum using quartz sand as ammoxidation archaeal, and using quartz sand as the seed of passage, the loss of the ammoxidation archaeal in succeeding generations is greatly reduced, the ammoxidation archaeal largely cultivated can be transferred in fresh culture medium, hence it is evident that shorten the period of culture.Combination penetrates the stronger azithromycin of biomembrane ability and Ciprofloxacin, quickly inhibits and kills symbiotic bacteria present in biomembrane, so that the abundance of ammoxidation archaeal is reached 90% or more in a cultivation cycle.This method can obtain the ammoxidation archaeal enriched substance of high-purity within some months, hence it is evident that time needed for shortening ammoxidation archaeal enrichment culture and culture difficulty are laid a good foundation for the research and application of ammoxidation archaeal.
Description
Technical field
The present invention relates to the fast enrichings from environment of the principle based on orienting enriching and fast purifying
The method of Nitrosocosmicus category ammoxidation archaeal.
Background technique
Autotrophic ammonia oxidizing is the first step by ammonium oxidation at nitrate nitrogen, is the core of global nitrogen cycle.The biochemistry is anti-
It should be considered an ammonia oxidizing bacteria for origin self-deformation bacterium door for a long time to undertake.In recent years the study found that from odd archaeal door
(thaumarchaeota) ammoxidation archaeal can also carry out ammoxidation.First was just successfully isolated until 2005
Strain ammoxidation archaeal.Ammoxidation archaeal and total Chemical Measurement of ammonia oxidizing bacteria are not different, but ammoxidation archaeal is to ammonia nitrogen
It is more much higher than ammonia oxidizing bacteria with the affinity of oxygen, it is thus possible to be adapted to the habitat under various nutrition limitations.They
Habitat range is than much bigger.Ammoxidation archaeal plays an important role in global nitrogen cycle；However, their effect not yet as
Ammonia oxidizing bacteria is widely studied like that.Also almost without industrial production and in terms of be applied.This
There is very big relationship with the property for being difficult to cultivate of ammoxidation archaeal.The reason of hardly possible culture, essentially consists in its maximum specific growth rate
The inhibiting effect that low and low concentration ammonia and nitrite grow it.Although being already expired at present away from turning out first plant of ammoxidation archaeal
It has gone the time of more than ten years, but the bacterial strain for cultivating obtained ammoxidation archaeal is still very rare, this is because its cultivation cycle
Very long, the enriched substance that the ammoxidation archaeal of high-purity is usually obtained from environment needs 2~3 years.Its application value also receives
Cultivate the restriction of difficulty.Particularly, Nitrosocosmicus, which belongs to ammoxidation archaeal, has stronger ammonia nitrogen tolerance, has very
Big application value, if fast culture can go out ammoxidation archaeal from environment, this will be conducive to its scientific research and production significantly
Using.
Currently, inhibited using antibiotic or it is bactericidal be the enrichment of ammoxidation archaeal common method, but due to bacterium
The drug resistance of interaction or bacterial antibiotic between archaeal, so that the enrichment culture of high purity ammonia oxidation archaeal is different
It is often difficult.
Summary of the invention
For being difficult to obtain ammoxidation archaeal from natural environment, the problems such as incubation time is too long, the purpose of the present invention exists
In providing a kind of fast enriching Nitrosocosmicus from the environment method for belonging to ammoxidation archaeal.
The present invention is passed using quartz sand as the attachment medium of ammoxidation archaeal using quartz sand as seed
In generation, substantially reduces the time that ammoxidation archaeal is obtained from environment.In addition to this, combination penetrate biomembrane ability stronger Ah
Miramycin and Ciprofloxacin quickly inhibit and kill symbiotic bacteria present in biomembrane, make ammonia oxygen in a cultivation cycle
The abundance for changing archaeal reaches 90% or more.
The purpose of the invention is achieved by the following technical solution:
A method of fast enriching Nitrosocosmicus belongs to ammoxidation archaeal from environment, includes the following steps:
(1) ampicillin and kanamycins are added in ammoxidation archaeal enriched medium, is inoculated with sample, and add stone
Sand starts the orienting enriching culture of first stage as medium；
(2) when ammonia nitrogen concentration drops to initial concentration 20~30% (preferably 20%) in culture, quartz sand is collected
And it is transferred in fresh ammoxidation archaeal enriched medium and carries out secondary culture；
(3) in secondary culture, when ammoxidation archaeal 16S rDNA copy number reaches 10 in every gram of quartz sand7~108Copy
(preferably 108Copy) when, start the fast purifying of second stage, that is, collects quartz sand and be transferred to fresh ammoxidation archaeal richness
Collect in culture medium, adds azithromycin and Ciprofloxacin is Antibiotics combination, kill the bacterium on quartz sand in biomembrane and suppression
The growth of bacterium in system purifying incubation；
(4) evaluation of high purity ammonia oxidation archaeal.
Preferably, sample described in step (1) is pedotheque.
Preferably, the quartz sand is the quartz sand of 8~16 mesh.
The ammoxidation archaeal enriched medium are as follows: 1g/L NaCl, 0.4g/L MgCl2·6H2O、0.1g/L
CaCl2·2H2O、0.2g/L KH2PO4, 0.5g/L KCl, 1mL/L microelement (1.5g/L FeCl2·4H2O, 190mg/L
CoCl2·6H2O, 100mg/L MnCl2·6H2O, 70mg/L ZnCl2, 62mg/L HBO3, 36mg/L Na2MoO4·2H2O,
24mg/L NiCl2·6H2O, 17mg/L CuCl2·2H2O), 1mL/L Fe-EDTA solution (49.14g/L FeSO4·7H2O、
196.896g/L EDTANa2·4H2O)、336mg/L NaHCO3With 107mg/L NH4Cl, pH 7.0.
Concentration difference of the ampicillin and kanamycins that first stage uses in ammoxidation archaeal enriched medium
For 40~60mg/L (preferably 50mg/L), the quartz sand volume of addition is 10~15% (w/v) (preferably 10%), condition of culture
For 28~32 DEG C (preferably 30 DEG C) dark stationary cultures.
Concentration of the azithromycin and Ciprofloxacin that second stage uses in ammoxidation archaeal enriched medium is respectively
40~60mg/L (preferably 50mg/L).
The secondary culture is using quartz sand as seed, and each quartz sand topples over supernatant when collecting.
The evaluation of the ammoxidation archaeal uses absolute fluorescence quantitative PCR -- calibration curve method, and Nitrosocosmicus belongs to
Ammoxidation archaeal 16S rDNA specific primer are as follows:
SS16S-1F:5 '-GCGCGAAACCTCTGCAATAG-3 '；
SS16S-1R:5 '-CCCAATAAACGTCCCGACCA-3 '；
Belong to quantifying for ammoxidation archaeal, bacterial 16 S rDNA universal primer for Nitrosocosmicus are as follows:
1369F:5 '-CGGTGAATACGTTCYCGG-3 '；
1492R:5 '-GYYACCTTGTTACGACTT-3 '.
Specifically,
According to obtained ammoxidation archaeal CAWith bacterium CB16S rDNA copy number calculate ammoxidation archaeal abundance
PA:
Quartz sand quality is MS, the volume of the DNA solution of extraction is V1, for quantitative PCR reaction DNA solution volume be
V2, the quantity Q of ammoxidation archaeal on quartz sand is calculated with thisA:
Nitrosocosmicus belongs to ammoxidation archaeal with the characteristic for forming biomembrane in dielectric surface, and the present invention will be sharp
With the enrichment culture of ability the type ammoxidation archaeal of its film forming, a kind of novel ammoxidation archaeal enrichment strategy is illustrated,
And the enriched substance of high-purity is obtained in a short time.
The present invention has the following advantages and effects with respect to the prior art:
(1) attaching substratum of the quartz sand as ammoxidation archaeal is used, and using quartz sand as the seed of passage, significantly
The loss for reducing the ammoxidation archaeal in succeeding generations, relative to the currently used liquid strain with 10%~20% inoculum concentration
The ammoxidation archaeal largely cultivated can be transferred in fresh culture medium by passage mode, this method, hence it is evident that shorten training
The feeding period.
(2) it using the biomembrane of Ciprofloxacin and azithromycin processing ammoxidation archaeal, can significantly improve in a short time
The abundance of ammoxidation archaeal.If using both antibiotic in traditional training method, can not be obtained enough in the short time
The enriched substance of quantity and high abundance.
(3) training method is simple, on the basis of no increase technical difficulty, significantly shortens culture difficulty and time.
(4) this method can obtain the ammoxidation archaeal enriched substance of high-purity within some months, hence it is evident that shorten ammoxidation Gu
Time needed for bacterium enrichment culture lays a good foundation for the research and application of ammoxidation archaeal.
Detailed description of the invention
It is that the consumption of ammonia nitrogen and the accumulation of nitrite nitrogen are bent in the incubation of switching carrier that Fig. 1, which is with quartz sand,
Line.
Fig. 2 is with quartz sand be transfer carrier incubation in ammoxidation archaeal quantity and ratio variation.
Fig. 3 is the quantity and ratio of ammoxidation archaeal in enriched substance after adding Ciprofloxacin and azithromycin.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels
And material.
Embodiment one:
(1) in inoculation 5g soil to the triangular flask containing 100mL ammoxidation archaeal enriched medium, and 10g quartz is added
Sand (8~16 mesh), the formula of antibiotic are the kanamycins and ampicillin of 50mg/L, 30 DEG C of dark stationary cultures.Work as ammonia
After the concentration of nitrogen drops to initial concentration 20%, transfer quartz sand to fresh ammoxidation archaeal enriched medium detects for every 3 days
Ammonia nitrogen and nitrite nitrogen concentration in enriched substance.As a result as shown in Figure 1, with passage progress, consume culture medium in
The speed of ammonia nitrogen obviously accelerate to cultivate a generation Dai Shicong it is initial shortened within 24 days 6 days, this explanation is switching with quartz sand
Carrier can significantly accelerate the enrichment rate of ammoxidation archaeal.
(2) it is each be commissioned to train support after, extract quartz sand succeeding generations in DNA, every generation take 1.2g quartz sand use
It is extracted in DNA, DNA is diluted in 50 μ L TE buffers, takes 1 μ L DNA for fluorescent quantitative PCR experiment, it is ancient to calculate ammoxidation
The quantity and ratio of bacterium.From figure 2 it can be seen that ammoxidation archaeal was gradually increased in 0~78 day quantity, and reached at 78 days
9 × 107Every g quartz sand.Ammoxidation archaeal quantity does not obviously increase later, and quantity maintains a higher level.
It can be obtained by the enriched substance of the sufficiently high ammoxidation archaeal of a quantity in the time of this explanation 80 days or so.Ammonia oxygen after 52 days
The abundance for changing archaeal has reached 40%, maintains between 30~40% always in enrichment culture hereafter, illustrates the first stage
Enrichment fail obtain high abundance ammoxidation archaeal.
(3) quartz sand containing high strength ammonia oxidation archaeal of acquisition is inoculated into new ammoxidation archaeal enriched medium
In, the Ciprofloxacin and azithromycin of 50mg/L are added, after ammonia nitrogen runs out of in culture medium, takes 1.2g quartz sand for DNA
It extracts, DNA is diluted in 50 μ L TE buffers, is taken 1 μ L DNA for fluorescent quantitative PCR experiment, is calculated the number of ammoxidation archaeal
Amount and ratio.It can be found that the abundance of ammoxidation archaeal has risen to 90% by 40% or so before from Fig. 3, and
It was continuously cultivated under the action of Ciprofloxacin and azithromycin for 3 generations, the abundance of ammoxidation archaeal still may remain in 90% or more.
Illustrate the ammoxidation archaeal that the antibiotic method can effectively in purification enrichment object.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>South China Science & Engineering University
<120>a kind of fast enriching Nitrosocosmicus from environment belongs to the method for ammoxidation archaeal
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> SS16S-1F
<400> 1
gcgcgaaacc tctgcaatag 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> SS16S-1R
<400> 2
cccaataaac gtcccgacca 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> 1369F
<400> 3
cggtgaatac gttcycgg 18
<210> 4
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> 1492R
<400> 4
gyyaccttgt tacgactt 18
Claims (10)
1. a kind of fast enriching Nitrosocosmicus from environment belongs to the method for ammoxidation archaeal, it is characterised in that: including such as
Lower step:
(1) ampicillin and kanamycins are added in ammoxidation archaeal enriched medium, is inoculated with sample, and add quartz sand
As medium, start the orienting enriching culture of first stage；
(2) it when ammonia nitrogen concentration drops to initial concentration 20~30% in culture, collects quartz sand and is transferred to fresh ammoxidation
Secondary culture is carried out in archaeal enriched medium；
(3) in secondary culture, when ammoxidation archaeal 16S rDNA copy number reaches 10 in every gram of quartz sand7~108When copy, open
The fast purifying of beginning second stage, i.e. collection quartz sand are simultaneously transferred in fresh ammoxidation archaeal enriched medium, add Archie
Mycin and Ciprofloxacin are Antibiotics combination, kill the bacterium on quartz sand in biomembrane and inhibit to purify bacterium in incubation
Growth；
(4) evaluation of high purity ammonia oxidation archaeal.
2. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
Sample described in step (1) is pedotheque.
3. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
The quartz sand is the quartz sand of 8~16 mesh.
4. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
The ammoxidation archaeal enriched medium are as follows: 1g/L NaCl, 0.4g/L MgCl2·6H2O、0.1g/L CaCl2·
2H2O、0.2g/L KH2PO4, 0.5g/L KCl, 1mL/L microelement, 1mL/L Fe-EDTA solution, 336mg/L NaHCO3With
107mg/L NH4Cl, pH 7.0；
The microelement is 1.5g/L FeCl2·4H2O, 190mg/L CoCl2·6H2O, 100mg/L MnCl2·6H2O,
70mg/L ZnCl2, 62mg/L HBO3, 36mg/L Na2MoO4·2H2O, 24mg/L NiCl2·6H2O, 17mg/L CuCl2·
2H2O；
The Fe-EDTA solution is 49.14g/L FeSO4·7H2O、196.896g/L EDTANa2·4H2O。
5. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
Concentration of the ampicillin and kanamycins that first stage uses in ammoxidation archaeal enriched medium is respectively 40
~60mg/L, the quartz sand volume of addition are 10~15%w/v, and condition of culture is 28~32 DEG C of dark stationary cultures.
6. according to claim 1 or 5 from environment fast enriching Nitrosocosmicus belong to ammoxidation archaeal side
Method, it is characterised in that:
Concentration of the ampicillin and kanamycins that first stage uses in ammoxidation archaeal enriched medium is respectively
50mg/L, the quartz sand volume of addition are 10%w/v, and condition of culture is 30 DEG C of dark stationary cultures.
7. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
Concentration of the azithromycin and Ciprofloxacin that second stage uses in ammoxidation archaeal enriched medium is respectively 40~
60mg/L。
8. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
The secondary culture is using quartz sand as seed, and each quartz sand topples over supernatant when collecting.
9. the fast enriching Nitrosocosmicus according to claim 1 from environment belongs to the method for ammoxidation archaeal,
It is characterized in that:
The evaluation of the ammoxidation archaeal uses absolute fluorescence quantitative PCR -- calibration curve method, and Nitrosocosmicus belongs to ammonia oxygen
Change archaeal 16S rDNA specific primer are as follows:
SS16S-1F:5 '-GCGCGAAACCTCTGCAATAG-3 '；
SS16S-1R:5 '-CCCAATAAACGTCCCGACCA-3 '；
Belong to quantifying for ammoxidation archaeal, bacterial 16 S rDNA universal primer for Nitrosocosmicus are as follows:
1369F:5 '-CGGTGAATACGTTCYCGG-3 '；
1492R:5 '-GYYACCTTGTTACGACTT-3 '.
10. the fast enriching Nitrosocosmicus according to claim 9 from environment belongs to the method for ammoxidation archaeal,
It is characterized by:
According to obtained ammoxidation archaeal CAWith bacterium CB16S rDNA copy number calculate ammoxidation archaeal abundance PA:
Quartz sand quality is MS, the volume of the DNA solution of extraction is V1, the DNA solution volume for quantitative PCR reaction is V2, with
This calculates the quantity Q of ammoxidation archaeal on quartz sandA:
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Cited By (2)
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| CN113136386A (en) * | 2020-01-20 | 2021-07-20 | 上海市园林科学规划研究院 | Method for rapidly detecting effective manganese content in soil of urban relocation land by using archaea molecular marker OTU127 |
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| CN110616175A (en) * | 2019-10-25 | 2019-12-27 | 中国水产科学研究院渔业机械仪器研究所 | Ammonia oxidizing archaea and fresh water pond ammonia oxidizing archaea enrichment culture method |
| CN110616175B (en) * | 2019-10-25 | 2021-03-26 | 中国水产科学研究院渔业机械仪器研究所 | Ammonia oxidizing archaea and fresh water pond ammonia oxidizing archaea enrichment culture method |
| CN113136386A (en) * | 2020-01-20 | 2021-07-20 | 上海市园林科学规划研究院 | Method for rapidly detecting effective manganese content in soil of urban relocation land by using archaea molecular marker OTU127 |
| CN113136386B (en) * | 2020-01-20 | 2022-10-14 | 上海市园林科学规划研究院 | Method for rapidly detecting effective manganese content of soil in urban relocation land by using archaea molecular marker OTU127 |
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