CN108300666A - It is a kind of to prepare with determination15The N of N abundance2It O and is based on15The method that N tracer methods measure nitrogen cycle - Google Patents
It is a kind of to prepare with determination15The N of N abundance2It O and is based on15The method that N tracer methods measure nitrogen cycle Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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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
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/385—Assays involving biological materials from specific organisms or of a specific nature from fungi from Penicillium
Abstract
The present invention relates to a kind of prepared using penicillium janthinellum to have determination15The N of N abundance2It O and is based on15The method that N tracer methods measure nitrogen cycle, belongs to biotechnology.Penicillium janthinellum in the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 7th in August in 2017, and it is CGMCCNo.14146 to preserve number, has and determines15The N of N abundance2The preparation method of O includes the following steps：Pedotheque is acquired, the culture of bacterial strain is carried out after dilution, surveys air sifter choosing and identification；Using with determination15The NaNO of N abundance3Cha Shi fluid nutrient mediums are configured, and are inoculated with microassembly robot bacteria strain, the N of generation is collected after shaken cultivation2O is added lye and absorbs removing CO therein2To get to determination15The N of N abundance2O.The method aerogenesis of the present invention is quick, accurate and easy to operate, at low cost；Gas obtained is used for the research of Nitrogen Cycling as calibrating gas, and result precision and accuracy are high.
Description
Technical field
The invention belongs to biotechnologies, and being related to a kind of prepared using penicillium janthinellum has determination15The N of N abundance2O
And it is based on15The method that N tracer methods measure nitrogen cycle.
Background technology
15N isotope tracer techniques are that we carry out the research most effectual way of Nitrogen Cycling, it can distinguish nitrogen and respectively have enough to meet the need
The rate and flux of process (mineralising, nitrification, denitrification etc.).At present for15NO3 -- N and15NO2 -The detection of-N is to pass through addition
Chemical reagent (such as hydroxylamine hydrochloride), nitrate nitrogen and nitrite nitrogen are reduced into15N2O gases are measured.In this method, it needs
With standard15N2O gases are calibrated.And it is current15N2The preparation method of O mainly will be with15N label nitrite be
Raw material is generated by chemical reduction reaction.But there are larger problems for the method：First, nitrite is unstable, during storage
It is easily oxidized to nitrate, is influenced15N2The content and abundance of O；Second is that generate15N2O gases are not easy to maintain, can only be now with existing
With.
N can also be generated by the denitrification of microorganism2O, for a long time, the mankind think always only bacterium ability
Anti-nitration reaction is carried out, the product of Bacterial Denitrification at One Time effect is mainly N2, however in 1972, the mankind have found Basidiomycota for the first time
There is also denitrification activities in Ascomycota, it was found that it is a variety of true that some are previously considered to stringent aerobic Fusarium oxysporum etc.
Bacterium can also carry out denitrification.Fungi is a kind of eucaryote, has increasingly complex structure compared with bacterium, is Soil Nitrogen
One of important participant in cyclic process.Compared to the denitrification of bacterium, the unique distinction of these fungies is that fungi
In there are P450 (Cytochrome P450, nitric oxidereductase), using NADH (reducibility coenzyme I) or NADPH (reduction
Property Coenzyme I I) as direct electron donor NO is reduced to N2O.The research about fungi denitrification focuses mostly in true at present
Bacterium is to soil N2The contribution of O releases, yet there are no to prepare using fungi production has determining abundance15N2The research of O.
Invention content
The purpose of the present invention is in view of the above-mentioned problems existing in the prior art, it is proposed that and it is a kind of to utilize penicillium janthinellum, lead to
Crossing addition has determination15The NaNO of N abundance3For substrate, determined to fast and accurately prepare to have15The N of N abundance2O gases,
It is easy to operate, cost economy.
Object of the invention can be realized by the following technical scheme：
A kind of penicillium janthinellum (Penicillium janthinellum) bacterial strain, is preserved in August in 2017 on the 7th
China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is CGMCC No.14146 (preservation to preserve number
Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode：100101, phone：
010-64807355, Email：cgmcc@sun.im.ac.cn).
Another object of the present invention is to provide a kind of prepared using the penicillium janthinellum to have determination15N abundance
N2The method of O, described method includes following steps：
S1, bacterial screening：Pedotheque is acquired, the culture of bacterial strain is carried out after dilution, surveys air sifter choosing and identification；
S2, strain confirm：The microassembly robot bacteria strain that screening obtains is inoculated into respectively with a series of differences15N abundance
NaNO3Cha Shi fluid nutrient mediums in, the N of generation is collected after shaken cultivation2O is added lye and absorbs removing CO therein2, use
Isotope mass spectrometer measures N2O's15N abundance, and standard curve is drawn, standard curve is linearly good, shows micro- in step S1
Purple Penicillium notatum, which can be used for preparing having, to be determined15The N of N abundance2O；
S3, there is determination15The N of N abundance2The preparation of O：Using with determination15The NaNO of N abundance3Configure the training of Cha Shi liquid
Base, and the microassembly robot bacteria strain in inoculation step S2 are supported, the N of generation is collected after shaken cultivation2O is added lye and absorbs removing
CO therein2To get to determination15The N of N abundance2O。
Preferably, the pedotheque comes from Hangzhou Dragon Well tea tea place.
Preferably, described be diluted to pedotheque being diluted to 10 using deionized water classification-5Times.
Preferably, the diluted detailed process of classification is to take Hangzhou Dragon Well tea tea garden soil sample 5g to after sterilizing
100mL triangular flasks in, 45mL sterile deionized waters are added, to obtain soil suspension be 10 to vortex concussion 1min-1It dilutes again
Liquid；Then 4 test tubes of 9mL sterile deionized waters will be filled respectively by 10-2To 10-5Sequence be numbered, with liquid relief test tube
Draw 1ml 10-1Times dilution to number is 10-2Test tube in, gently pressure-vaccum three times, makes suspension mix well, and obtains 10-2Times
Dilution；And so on, number 10 is completed in repetitive displacement, mixing operation-3To 10-5Test tube dilution, obtain 10-5It dilutes again
Liquid.
Preferably, the culture of bacterial strain is that the pedotheque after dilution is coated on Cha Shi solid cultures in the step S1
On base, isolates fungi single bacterium colony and be inoculated into Cha Shi fluid nutrient mediums, shaken cultivation.
Preferably, the temperature of shaken cultivation is 28~32 DEG C in the step S1, rotating speed is 150~200rpm.
Preferably, the detailed process for surveying air sifter choosing in the step S1 is, respectively in shaken cultivation 1, after 3,7 days, to make
It is measured with greenhouse gases determining instrument and generates N2The content of O gases, screening obtain N2The higher bacterial strain of O gas contents.
Preferably, the strain obtained to screening according to flat-plate bacterial colony form and ITS sequencings that is accredited as is identified.
The present invention utilizes the penicillium janthinellum screened from soil, has by addition and determines15The NaNO of N abundance3
For substrate, can fast and accurately prepare with determination15The N of N abundance2O gases, easy to operate, cost economy.In the present invention
Penicillium janthinellum (Penicillium janthinellum) is a kind of production N2O fungies derive from Hangzhou Dragon Well tea tea garden soil sample
Product, by detaching, surveying gas, purifying screening obtains, and there is fold on bacterium colony surface, and mycelia just gradually becomes lavender for white, mitogenetic
Sporophore top forms the branch of broom shape, and conidium ellipse, surface is smooth, can pass through using sodium nitrate as nitrogen source substrate
Denitrification generates N2O, and there is higher gas production.The present invention confirms that the strain that screening obtains exists using calibration curve method
It is different15Whether can be completely by NaNO under N abundance3In15N is fully converted to15N2O, after measured, standard curve are linearly good,
It screens obtained penicillium janthinellum and is suitable for production with determination15The N of N abundance2O。
Preferably, the microassembly robot bacteria strain screened in step S1 and S2 is protected in 0~5 DEG C of lower inclined plane refrigeration
It deposits.In use, by activated growth in stored refrigerated penicillium janthinellum inoculation to solid plate, expand at 28~32 DEG C
Culture 2.5~3.5 days.
Preferably, the Cha Shi solid mediums include the ingredient of following content：NaNO31.8~2.2g, K2HPO4
0.8~1.3g, KCl 0.4~0.6g, MgSO40.4~0.6g, FeSO40.01g, 27~32g of sucrose, 15~20g of agar, water
Sterilize after the completion of the preparation of 1000ml, Cha Shi solid medium at 115~126 DEG C 17~23min.
Preferably, the Cha Shi fluid nutrient mediums include the ingredient of following content：NaNO31.8~2.2g, K2HPO4
0.8~1.2g, KCl 0.4~0.6g, MgSO40.4~0.6g, FeSO40.01g, 27~32g of sucrose, water 1000ml, Cha Shi
Sterilize after the completion of fluid nutrient medium preparation at 115~126 DEG C 17~23min.
Preferably, the process of the step S2 and the shaken cultivation in step S3 is, at 25~32 DEG C with 150~
200rpm speed oscillations culture 2.5~3.5 days.
Preferably, the NaOH solution that the lye in the step S2 and step S3 is 1.5~2.5mol/L.
The third object of the present invention is that providing one kind is based on15The method that N tracer methods measure nitrogen cycle, follows nitrogen
During ring15NO3 -- N and15NO2 -- N is detected, and uses the method as described in any claim in claim 2~8
Having for preparing determines15The N of N abundance2O is that calibrating gas calibrates testing result.
Compared with prior art, the invention has the advantages that：The penicillium janthinellum obtained using screening, to have
It determines15The NaNO of N abundance3For substrate, obtaining has determination15The N of N abundance2O gases, aerogenesis quickly, it is accurate and easy to operate,
At low cost, aerogenesis fungi can reuse, and energy consumption is low, reduces gas storage and transport cost, can prepare tool as needed
There is determination15The N of N abundance2O gases；The determination prepared using the present invention15The N of N abundance2O gases are used for nitrogen as calibrating gas
The research of cycle, during Nitrogen Cycling15NO3 -With15NO2 -In nitrogen carry out qualitative and quantitative detection, avoid tradition
It is prepared using oxidation-reduction method15N2O raw material de-stabilising effects15N2The problem of content and abundance of O, result precision and accurate
Degree is high.
Description of the drawings
Fig. 1 is difference15N abundance NaNO3The N of generation2O's15The standard curve of N abundance.
Specific implementation mode
The following is specific embodiments of the present invention, and technical scheme of the present invention will be further described, but the present invention is simultaneously
It is not limited to these embodiments.
Embodiment 1
(1), it is prepared according to following component composition and looks into formula solid medium：NaNO32g, K2HPO41g, KCl 0.5g,
MgSO40.5g, FeSO40.01g, sucrose 30g, agar 18g, water 1000ml, sterilize after the completion of preparation at 121 DEG C 20min；
It is prepared according to following component composition and looks into formula fluid nutrient medium：NaNO32g, K2HPO41g, KCl 0.5g, MgSO4
0.5g, FeSO40.01g, sucrose 30g, water 1000ml, sterilize after the completion of preparation at 121 DEG C 20min.
(2) bacterial screening：
It acquires in 100mL triangular flasks of the Hangzhou Dragon Well tea tea garden soil sample 5g to after sterilizing, 45mL sterilizing deionizations is added
Water, it is 10 that vortex concussion 1min, which obtains soil suspension,-1Times dilution；Then 9mL sterile deionized waters will be filled respectively
4 test tubes press 10-2To 10-5Sequence be numbered, with liquid relief test tube draw 1ml 10-1Times dilution to number is 10-2's
In test tube, gently pressure-vaccum three times, makes suspension mix well, and obtains 10-2Times dilution；And so on, repetitive displacement mixing behaviour
Make, completes number 10-3To 10-5Test tube dilution, obtain 10-5Times dilution；
By 10-5Times dilution is coated on Cha Shi solid mediums, is isolated fungi single bacterium colony and is inoculated into Cha Shi liquid
In culture medium, with 150~200rpm speed oscillation cultures at 30 DEG C, respectively in shaken cultivation 1, after 3,7 days, greenhouse gas is used
Body measurement Instrument measuring generates N2The content of O gases, screening obtain N2The higher strain of O gas contents, further according to flat-plate bacterial colony shape
The strain that state and ITS sequencings obtain screening is identified, penicillium janthinellum is identified as；
The penicillium janthinellum that screening is obtained is stored refrigerated in 4 DEG C of lower inclined planes.
(3), strain confirms：
Stored refrigerated penicillium janthinellum is inoculated into activated growth on solid plate, expands culture 3 days at 30 DEG C,
Then penicillium janthinellum is inoculated into respectively with 0%, 1%, 2%, 5%15N abundance NaNO3Cha Shi fluid nutrient mediums in,
Collect the N of generation after 3 days with 180rpm speed oscillations culture at 28 DEG C2The NaOH solution of 5mL 2.0mol/L is added in O.
It absorbs and removes CO therein2, N is measured using isotope mass spectrometer2O's15N abundance, measurement result is as shown in table 1, and draws mark
Directrix curve (as shown in Figure 1), as shown in Figure 1, the linear good (R of standard curve2=1), illustrate the penicillium janthinellum in step S1
It can prepare with determination15The N of N abundance2O, since there are micro in the Nature15N, therefore work as NaNO3's15N abundance is 0
When, the N of generation2O's15N abundance is not 0.
Table 1：It is different15N abundance NaNO3The N of generation2O's15N abundance
| NaNO3's15N abundance | N2O's15N abundance |
| 0 | 0.3645% |
| 1% | 1.3066% |
| 2% | 2.2265% |
| 5% | 5.006% |
(4), have and determine15The N of N abundance2The preparation of O：
As needed using with determination15The NaNO of N abundance3Cha Shi fluid nutrient mediums are configured, and are inoculated with penicillium janthinellum,
Collect the N of generation after 3 days with 180rpm speed oscillations culture at 28 DEG C2O, the NaOH solution that 5mL2.0mol/L is added absorb
Remove CO therein2To get to determination15The N of N abundance2O。
Embodiment 2
The present embodiment is differed only in embodiment 1, look into formula solid medium at being grouped into：NaNO31.8g
K2HPO40.8g, KCl 0.4g, MgSO40.4g, FeSO40.01g, sucrose 27g, agar 20g, water 1000ml are prepared and are completed
Sterilize at 126 DEG C 17min afterwards；
Look into formula fluid nutrient medium at being grouped into：NaNO31.8g, K2HPO40.8g, KCl 0.4g, MgSO4
0.4g, FeSO40.01g, sucrose 27g, water 1000ml, sterilize after the completion of preparation at 126 DEG C 17min.
Embodiment 3
The present embodiment is differed only in embodiment 1, look into formula solid medium at being grouped into：NaNO32.2g
K2HPO41.3g, KCl 0.6g, MgSO40.6g, FeSO40.01g, sucrose 32g, agar 15g, water 1000ml are prepared and are completed
Sterilize at 115 DEG C 23min afterwards；
Look into formula fluid nutrient medium at being grouped into：NaNO32.2g, K2HPO41.3g, KCl 0.6g, MgSO4
0.6g, FeSO40.01g, sucrose 32g, water 1000ml, sterilize after the completion of preparation at 115 DEG C 23min.
Embodiment 4
The present embodiment is differed only in embodiment 1, and the temperature of shaken cultivation is 28 DEG C during bacterial screening, rotating speed
For 200rpm.
Embodiment 5
The present embodiment is differed only in embodiment 1, and the temperature of shaken cultivation is 32 DEG C during bacterial screening, rotating speed
For 150rpm.
Embodiment 6
The present embodiment is differed only in embodiment 1, and bacterial screening screens obtained penicillium janthinellum refrigeration in the process
The temperature of preservation is 0 DEG C.
Embodiment 7
The present embodiment is differed only in embodiment 1, and bacterial screening screens obtained penicillium janthinellum refrigeration in the process
The temperature of preservation is 5 DEG C.
Embodiment 8
In the step of the present embodiment is differed only in embodiment 1, and strain confirms, the temperature for expanding culture is 28 DEG C,
Incubation time is 3.5 days, and the temperature of shaken cultivation is 25 DEG C, and incubation time is 3.5 days, rotating speed 200rpm.
Embodiment 9
In the step of the present embodiment is differed only in embodiment 1, and strain confirms, the temperature for expanding culture is 32 DEG C,
Incubation time is 2.5 days, and the temperature of shaken cultivation is 32 DEG C, and incubation time is 2.5 days, rotating speed 150rpm.
Embodiment 10
The present embodiment is differed only in embodiment 1, is had and is determined15The N of N abundance2In the preparation process of O, oscillation training
Foster temperature is 25 DEG C, and the time is 3.5 days, rotating speed 150rpm.
Embodiment 11
The present embodiment is differed only in embodiment 1, is had and is determined15The N of N abundance2In the preparation process of O, oscillation training
Foster temperature is 32 DEG C, and the time is 2.5 days, rotating speed 200rpm.
Embodiment 12
The present embodiment is differed only in embodiment 1, a concentration of 1.5mol/L of NaOH solution.
Embodiment 13
The present embodiment is differed only in embodiment 1, a concentration of 2.5mol/L of NaOH solution.
Embodiment 14
During Nitrogen Cycling15NO3 -- N and15NO2 -- N is detected, and true using having for being prepared in embodiment 1
It is fixed15The N of N abundance2O is that calibrating gas calibrates testing result, carries out 5 parallel tests respectively.
Comparative example 1
Using15The nitrite of N labels is the N that raw material is generated by chemical reduction reaction2O is that calibrating gas ties detection
Fruit is calibrated, and carries out 5 parallel tests respectively.
The penicillium janthinellum that the present invention screens, aerogenesis is quick, accurate, the penicillium janthinellum in the embodiment 1 of use,
It is calculated as according to the dry weight of fungi：7 days every gram of dry weights of culture produce N2O gases add up as 57.2g/L；It calculates, connects according to serum bottle
Kind penicillium janthinellum measured its N in liquid czapek's medium at the 1 of culture, 3,7 day2O burst sizes, the results are shown in table below：
| Cultivated days | Every bottle of N2O burst sizes mg/L |
| 0 | 0 |
| 1 | 34 |
| 3 | 740 |
| 7 | 6858 |
Comparing embodiment 14 and comparative example 1, the deviation between testing result and true value in embodiment 14 are respectively less than 0.01 μ
G, precision CV are less than 0.7%, and the deviation between the testing result and true value of comparative example 1 is 0.02-0.06 μ g, precision CV
More than 0.8%
In conclusion in order to avoid tradition is prepared using chemical method15N2Raw material is unstable during O, and product is caused to contain
Amount and the inaccurate and not easy to maintain problem of abundance, creativeness use the production N filtered out2O amounts big penicillium janthinellums carries out15N2The preparation of O, easy to operate quick, at low cost, N obtained2O has determining15N abundance is had using prepared by the present invention
It determines15The N of N abundance2O as calibrating gas to Nitrogen Cycling during15NO3 -- N and15NO2 -The testing result of-N carries out school
Standard, the calibrating gas that accuracy and precision is prepared compared with conventional oxidation reduction method significantly improve, are conducive in scientific research
Obtain accurate conclusion.
Specific embodiment described herein is only an example for the spirit of the invention.Technology belonging to the present invention is led
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (9)
1. a kind of penicillium janthinellum (Penicillium janthinellum) bacterial strain, is preserved in China on 7th in August in 2017
Microbiological Culture Collection administration committee common micro-organisms center, it is CGMCC No.14146 to preserve number.
2. a kind of prepared using penicillium janthinellum described in claim 1 has determination15The N of N abundance2The method of O, feature exist
In described method includes following steps：
S1, bacterial screening：Pedotheque is acquired, the culture of bacterial strain is carried out after dilution, surveys air sifter choosing and identification；
S2, strain confirm：The microassembly robot bacteria strain that screening obtains is inoculated into respectively with a series of differences15N abundance NaNO3
Cha Shi fluid nutrient mediums in, the N of generation is collected after shaken cultivation2O is added lye and absorbs removing CO therein2, use same position
Quality spectrometer measures N2O's15N abundance, and standard curve is drawn, standard curve is linearly good, shows micro- purple green in step S1
Mould, which can be used for preparing having, to be determined15The N of N abundance2O；
S3, there is determination15The N of N abundance2The preparation of O：Using with determination15The NaNO of N abundance3Cha Shi fluid nutrient mediums are configured,
And the microassembly robot bacteria strain in inoculation step S2, the N of generation is collected after shaken cultivation2It is therein that lye absorption removing is added in O
CO2To get to determination15The N of N abundance2O。
3. according to the method described in claim 2, it is characterized in that, in the step S1 culture of bacterial strain be the soil after diluting
Earth sample is coated on Cha Shi solid mediums, is isolated fungi single bacterium colony and is inoculated into Cha Shi fluid nutrient mediums, oscillation training
It supports.
4. according to the method described in claim 3, it is characterized in that, the temperature of shaken cultivation is 28~32 in the step S1
DEG C, rotating speed is 150~200rpm.
5. according to the method in claim 2 or 3, which is characterized in that the Cha Shi solid mediums include following content
Ingredient：NaNO31.8~2.2g, K2HPO40.8~1.3g, KCl 0.4~0.6g, MgSO40.4~0.6g, FeSO4
0.01g, 27~32g of sucrose, 15~20g of agar, after the completion of the preparation of water 1000ml, Cha Shi solid medium at 115~126 DEG C
Sterilize 17~23min.
6. according to the method in claim 2 or 3, which is characterized in that the Cha Shi fluid nutrient mediums include following content
Ingredient：NaNO31.8~2.2g, K2HPO40.8~1.2g, KCl 0.4~0.6g, MgSO40.4~0.6g, FeSO4
0.01g, 27~32g of sucrose, water 1000ml, Cha Shi fluid nutrient medium prepare after the completion of at 115~126 DEG C sterilizing 17~
23min。
7. according to the method described in claim 2, it is characterized in that, the temperature of the step S2 and the shaken cultivation in step S3
It it is 25~32 DEG C, rotating speed is 150~200rpm, and incubation time is 2.5~3.5 days.
8. according to the method described in claim 2, it is characterized in that, lye in the step S2 and step S3 be 1.5~
The NaOH solution of 2.5mol/L.
9. one kind is based on15The method that N tracer methods measure nitrogen cycle, which is characterized in that during nitrogen cycle15NO3 -- N and15NO2 -- N is detected, and is had using prepared by the method as described in any claim in claim 2~8
It determines15The N of N abundance2O is that calibrating gas calibrates testing result.
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|---|---|---|---|
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| CN201711385580.0A CN108300666B (en) | 2017-12-20 | 2017-12-20 | Method for preparing N2O with determined 15N abundance and measuring nitrogen circulation based on 15N isotope tracing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849787A (en) * | 2020-08-04 | 2020-10-30 | 安徽工业大学 | Endophytic fungus and application thereof in dye pollutant treatment |
| CN114350763A (en) * | 2021-12-03 | 2022-04-15 | 中国科学院城市环境研究所 | Method for identifying nitrifying microorganisms |
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| CN111849787B (en) * | 2020-08-04 | 2021-09-07 | 安徽工业大学 | Endophytic fungus and application thereof in dye pollutant treatment |
| CN114350763A (en) * | 2021-12-03 | 2022-04-15 | 中国科学院城市环境研究所 | Method for identifying nitrifying microorganisms |
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| CN108300666B (en) | 2021-10-22 |
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