CN108300666B - A method for preparing N2O with definite 15N abundance and measuring nitrogen cycle based on 15N isotope tracing method - Google Patents
A method for preparing N2O with definite 15N abundance and measuring nitrogen cycle based on 15N isotope tracing method Download PDFInfo
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Abstract
本发明涉及一种利用微紫青霉菌制备具有确定15N丰度的N2O及基于15N同位素示踪法测量氮循环的方法，属于生物技术领域。本发明中的微紫青霉菌已于2017年8月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保存编号为CGMCCNo.14146，具有确定15N丰度的N2O的制备方法包括如下步骤：采集土壤样品，稀释后进行菌株的培养、测气筛选和鉴定；使用具有确定15N丰度的NaNO3配置察氏液体培养基，并接种微紫青霉菌菌株，振荡培养后收集产生的N2O，加入碱液吸收除去其中的CO2，即得到具有确定15N丰度的N2O。本发明的方法产气快速、准确，且操作简便、成本低；制得的气体作为标准气体用于氮素循环的研究，结果准确度和精确度高。
The invention relates to a method for preparing N 2 O with a determined 15 N abundance by using Penicillium micropurpuri and a method for measuring nitrogen cycle based on a 15 N isotope tracing method, belonging to the field of biotechnology. The Penicillium micropurpuri in the present invention has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on August 7, 2017, and the preservation number is CGMCC No. 14146. The preparation method of N 2 O with a determined 15 N abundance includes the following steps: The steps are as follows: collecting soil samples, diluting them, and performing strain culture, gas measurement screening and identification; using NaNO 3 with a definite 15 N abundance to configure a Chapella liquid medium, and inoculating Penicillium micropurpuri strains, and collecting and producing after shaking culture The N 2 O is added to the alkaline solution to absorb and remove the CO 2 , that is, N 2 O with a definite 15 N abundance is obtained. The method of the invention produces gas quickly and accurately, and has the advantages of simple operation and low cost; the prepared gas is used as a standard gas for the research of nitrogen cycle, and the result has high accuracy and precision.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for preparing a microorganism with definite property by utilizing penicillium janthinillum15N abundance of N2O and based on15A method for measuring nitrogen circulation by an N isotope tracer method.
Background
15The N isotope tracer technique is the most effective method for researching nitrogen circulation, and can distinguish the speed and flux of each turnover process (mineralization, nitration, denitrification, etc.) of nitrogen. At present for15NO3 --N and15NO2 -detection of N by reduction of nitrate and nitrite nitrogen to form nitrate nitrogen by addition of a chemical reagent (e.g. hydroxylamine hydrochloride)15N2And measuring the O gas. In this method, it is necessary to use the standard15N2O gas was calibrated. At present, however, the15N2The preparation method of O mainly comprises the following steps15The nitrite marked by N is used as a raw material and is generated through chemical reduction reaction. However, this method has a major problem: firstly, nitrite is unstable and is easily oxidized into nitrate in the storage process15N2The content and abundance of O; secondly produced15N2The O gas is not easy to store and can only be prepared for use.
N can also be produced by denitrification of microorganisms2O, it has long been thought by man that denitrification can only be carried out by bacteria, the products of which are mainly N2However, in 1972, it was first discovered that denitrification activity was also present in Basidiomycota and Ascomycota, and that denitrification was also performed by a number of fungi such as Fusarium oxysporum, which was previously considered strictly aerobic. Fungi are eukaryotes, have a more complex structure than bacteria, and are among the important participants in the soil nitrogen cycle. These fungi are distinguished by the presence of P450 (cytochrome P450, nitric oxide reductase) in the fungus, compared to bacterial denitrification, and by the fact that NADH (reducing coenzyme I) or NADPH (reducing coenzyme II) can be used as direct electron donor for the reduction of NO to N2And O. The current research on the denitrification of fungi mostly focuses on the soil N caused by the fungi2Contribution to O release, no known contribution has been found to the production of compounds of defined abundance by fungal production15N2And (4) researching O.
Disclosure of Invention
The purpose of the invention is to solve the existing problemsThe above problems in the art have been solved by providing a microorganism having a specific activity using Penicillium janthinellum15N-abundant NaNO3Is a substrate, thereby rapidly and accurately preparing the product with determination15N abundance of N2O gas, simple operation and low cost.
The purpose of the invention can be realized by the following technical scheme:
a Penicillium janthinillum strain is preserved in China general microbiological culture Collection center at 8.7.2017, with the preservation number of CGMCC No.14146 (the preservation address: No. 3 of West Lu No.1 of Beijing Indoron, institute of microbiology of China academy of sciences, postal code: 100101, telephone: 010 + 64807355, electronic mail: CGMCC @ sun. im. ac. cn).
Another object of the present invention is to provide a method for preparing a microorganism having a definite property by using the Penicillium janthinellum15N abundance of N2O, said method comprising the steps of:
s1, strain screening: collecting a soil sample, diluting, and then culturing, testing gas, screening and identifying strains;
s2, strain confirmation: respectively inoculating the screened penicillium janthinillum strains to a strain with a series of differences15N abundance NaNO3In the Czochralski liquid medium, collecting the produced N after the shaking culture2Adding alkali liquor to absorb and remove CO in the O2Determination of N by isotope mass spectrometer2Of O15N abundance and drawing a standard curve with good linearity, which indicates that the penicillium janthinillum in the step S1 can be used for preparing the penicillium janthinillum with definite property15N abundance of N2O；
S3, having determination15N abundance of N2Preparation of O: using has determination15N-abundant NaNO3Preparing a Czochralski liquid culture medium, inoculating the penicillium janthinillum strain obtained in the step S2, performing shake culture, and collecting the generated N2Adding alkali liquor to absorb and remove CO in the O2That is, the determination of15N abundance of N2O。
Preferably, the soil sample is from the hang state dragon well tea garden.
Preferably, the dilution is to fractionally dilute the soil sample to 10 with deionized water-5And (4) doubling.
Preferably, the specific process of fractional dilution is that 5g of the Hangzhou Longjing tea garden soil sample is adopted to be added into a sterilized 100mL triangular flask, 45mL of sterilized deionized water is added, and vortex oscillation is carried out for 1min to obtain a soil suspension which is 10-1A double diluent; then, respectively putting 4 test tubes containing 9mL of sterilized deionized water according to the weight ratio of 10-2To 10-5Is numbered, 1ml of 10 is pipetted into a pipette tube-1Number of dilution is 10-2Gently blowing and sucking for three times in the test tube to fully mix the suspension to obtain 10-2A double diluent; repeating the transferring and mixing operations by analogy to finish the numbering 10-3To 10-5Dilution of the test tube of (2) to give 10-5Double dilution.
Preferably, the strain is cultured in step S1 by spreading the diluted soil sample on a czochralski solid medium, isolating a single fungal colony, inoculating the single fungal colony to a czochralski liquid medium, and culturing the single fungal colony with shaking.
Preferably, the temperature of the shaking culture in the step S1 is 28-32 ℃, and the rotation speed is 150-200 rpm.
Preferably, the step S1 of screening by measuring gas is to measure N production by using a greenhouse gas measuring instrument after shaking culture for 1,3 and 7 days, respectively2The content of O gas is screened to obtain N2The strain with higher O gas content.
Preferably, said identification identifies the screened species based on plate colony morphology and ITS sequencing.
The invention utilizes penicillium janthinillum screened from soil and has definite property by adding15N-abundant NaNO3Can be used as substrate for rapid and accurate preparation15N abundance of N2O gas, simple operation and low cost. The Penicillium janthinillum (Penicillium janthinellum) of the invention is a product for producing N2O fungus from HangzhouA soil sample of the Longjing tea garden is obtained through separation, gas measurement and purification screening, the surface of a bacterial colony has folds, hyphae are white and gradually change into light purple, broom-shaped branches are formed at the tops of conidiophores, the conidiophores are oval, the surface is smooth, sodium nitrate can be used as a nitrogen source substrate, and N is generated through denitrification2O, and has higher gas production. The invention adopts a standard curve method to confirm that the strains obtained by screening are different15Whether NaNO can be completely converted under N abundance3In (1)15Complete conversion of N into15N2O, the standard curve is good in linearity after determination, and the screened penicillium janthinillum is suitable for production and has definite performance15N abundance of N2O。
Preferably, the Penicillium janthinellum strains screened in the steps S1 and S2 are stored in a slant refrigerator at 0-5 ℃. When the strain is used, the refrigerated penicillium microphyllum strain is inoculated on a solid plate for activated growth, and is subjected to amplification culture at the temperature of 28-32 ℃ for 2.5-3.5 days.
Preferably, the Chaudhuri solid culture medium comprises the following components in percentage by weight: NaNO31.8～2.2g，K2HPO40.8～1.3g，KCl 0.4～0.6g，MgSO4 0.4～0.6g，FeSO40.01g, 27-32 g of cane sugar, 15-20 g of agar and 1000ml of water, and sterilizing for 17-23 min at 115-126 ℃ after the preparation of the Chaudhur solid culture medium is finished.
Preferably, the Chaudhuri liquid culture medium comprises the following components in percentage by weight: NaNO31.8～2.2g，K2HPO40.8～1.2g，KCl 0.4～0.6g，MgSO4 0.4～0.6g，FeSO40.01g, 27-32 g of cane sugar and 1000ml of water, and sterilizing for 17-23 min at 115-126 ℃ after the preparation of the Chaudhur liquid culture medium is finished.
Preferably, the shaking culture in the steps S2 and S3 is performed at a temperature of 25-32 ℃ and a rotation speed of 150-200 rpm for 2.5-3.5 days.
Preferably, the alkali liquor in the step S2 and the step S3 is 1.5-2.5 mol/L NaOH solution.
The third purpose of the invention is to provide a method based on15N isotopeMethod for measuring nitrogen cycle by tracer method, method for measuring nitrogen cycle in nitrogen cycle process15NO3 --N and15NO2 --N is detected, having a defined value, prepared using a method according to any one of claims 2 to 815N abundance of N2And O is standard gas to calibrate the detection result.
Compared with the prior art, the invention has the following beneficial effects: using the screened penicillium janthinillum to have confirmation15N-abundant NaNO3For the substrate, the obtained has a definition15N abundance of N2O gas, fast and accurate gas production, simple operation, low cost, reusable gas producing fungus, low energy consumption, reduced gas storage and transportation cost, and capability of being prepared according to requirements15N abundance of N2O gas; determination by the preparation of the invention15N abundance of N2The O gas is used as standard gas for researching nitrogen circulation in the nitrogen circulation process15NO3 -And15NO2 -the nitrogen element in the product is qualitatively and quantitatively detected, and the traditional preparation method using oxidation-reduction method is avoided15N2Influence of instability of O feedstock15N2The content and abundance of O, and the accuracy and precision of the result are high.
Drawings
FIG. 1 is a schematic diagram of15N abundance NaNO3Generation of N2Of O15Standard curve of N abundance.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.
Example 1
(1) Preparing a Chazuke solid culture medium according to the following components: NaNO3 2g，K2HPO4 1g，KCl 0.5g，MgSO4 0.5g，FeSO40.01g, 30g of cane sugar, 18g of agar and 1000ml of water, and sterilizing for 20min at 121 ℃ after preparation;
according toPreparing a check type liquid culture medium from the following components: NaNO3 2g，K2HPO4 1g，KCl 0.5g，MgSO40.5g，FeSO40.01g, 30g of cane sugar and 1000ml of water, and sterilizing for 20min at 121 ℃ after preparation.
(2) Strain screening:
collecting 5g of Hangzhou Longjing tea garden soil sample to a sterilized 100mL triangular flask, adding 45mL of sterilized deionized water, and performing vortex oscillation for 1min to obtain a soil suspension which is 10-1A double diluent; then, respectively putting 4 test tubes containing 9mL of sterilized deionized water according to the weight ratio of 10-2To 10-5Is numbered, 1ml of 10 is pipetted into a pipette tube-1Number of dilution is 10-2Gently blowing and sucking for three times in the test tube to fully mix the suspension to obtain 10-2A double diluent; repeating the transfer and mixing operation by analogy, and finishing the numbering 10-3To 10-5Dilution of the test tube of (2) to give 10-5A double diluent;
will 10-5Coating the diluted solution on a Chaudou solid culture medium, separating out a fungus single colony, inoculating the fungus single colony into a Chaudou liquid culture medium, performing shake culture at the rotating speed of 150-200 rpm at the temperature of 30 ℃, performing shake culture for 1,3 and 7 days respectively, and measuring the generated N by using a greenhouse gas measuring instrument2The content of O gas is screened to obtain N2Identifying the strains with higher O gas content according to the colony morphology of the plate and ITS sequencing, and identifying the strains as penicillium janthinellum;
and (4) freezing and storing the screened penicillium janthinillum at the inclined surface at the temperature of 4 ℃.
(3) And strain confirmation:
inoculating refrigerated Penicillium janthinellum to solid plate for activating growth, performing amplification culture at 30 deg.C for 3 days, and respectively inoculating 0%, 1%, 2%, and 5% of Penicillium janthinellum15N abundance NaNO3In the Czochralski liquid medium, the produced N was collected after 3 days of shaking culture at 180rpm at 28 ℃2O, 5mL of a 2.0mol/L NaOH solution was added. Absorbing and removing CO in the waste gas2Determination of N by isotope mass spectrometer2Of O15The N abundance and the measurement results are shown in table 1, and a standard curve (shown in fig. 1) is plotted, and it is seen from fig. 1 that the standard curve is good in linearity (R)21), it is explained that penicillium janthinillum of step S1 can be prepared with certainty15N abundance of N2O, due to the trace amount of natural substances15N, thus when NaNO3Is/are as follows15N abundance of 0, produced2Of O15The N abundance was not 0.
Table 1: is different15N abundance NaNO3Generation of N2Of O15Abundance of N
| NaNO3Is/are as follows15Abundance of N | N2Of O15Abundance of |
| 0 | 0.3645％ |
| 1％ | 1.3066％ |
| 2％ | 2.2265％ |
| 5％ | 5.006％ |
(4) Having determination of15N abundance of N2Preparation of O:
using on-demand determinations15N-abundant NaNO3Preparing a Chao's liquid culture medium, inoculating Penicillium purpureum, and culturing at 28 deg.C under shaking at 180rpm for 3 daysPost-collection of the generated N2O, adding 5ml of 2.0mol/L NaOH solution to absorb and remove CO in the solution2That is, the determination of15N abundance of N2O。
Example 2
This example differs from example 1 only in that the composition of the griffonia simplicifolia solid medium is: NaNO3 1.8g，K2HPO4 0.8g，KCl 0.4g，MgSO4 0.4g，FeSO40.01g, 27g of cane sugar, 20g of agar and 1000ml of water, and sterilizing for 17min at 126 ℃ after preparation;
the formula-searching liquid culture medium comprises the following components: NaNO3 1.8g，K2HPO4 0.8g，KCl 0.4g，MgSO4 0.4g，FeSO40.01g, 27g of cane sugar and 1000ml of water, and sterilizing for 17min at 126 ℃ after preparation.
Example 3
This example differs from example 1 only in that the composition of the griffonia simplicifolia solid medium is: NaNO3 2.2g，K2HPO4 1.3g，KCl 0.6g，MgSO4 0.6g，FeSO40.01g, 32g of cane sugar, 15g of agar and 1000ml of water, and sterilizing for 23min at 115 ℃ after preparation;
the formula-searching liquid culture medium comprises the following components: NaNO3 2.2g，K2HPO4 1.3g，KCl 0.6g，MgSO4 0.6g，FeSO40.01g, 32g of cane sugar and 1000ml of water, and sterilizing for 23min at 115 ℃ after preparation.
Example 4
This example differs from example 1 only in that the temperature of the shaking culture during the seed culture selection was 28 ℃ and the rotation speed was 200 rpm.
Example 5
This example differs from example 1 only in that the temperature of the shaking culture during the seed culture selection was 32 ℃ and the rotation speed was 150 rpm.
Example 6
This example differs from example 1 only in that the temperature for cold storage of the penicillium janthinillum obtained by screening in the strain screening process was 0 ℃.
Example 7
This example differs from example 1 only in that the temperature for cold storage of the penicillium janthinillum obtained by screening in the strain screening process was 5 ℃.
Example 8
This example differs from example 1 only in that in the step of identifying the seed culture, the temperature for the scale-up culture was 28 ℃, the culture time was 3.5 days, the temperature for the shaking culture was 25 ℃, the culture time was 3.5 days, and the rotation speed was 200 rpm.
Example 9
This example differs from example 1 only in that in the step of identifying the seed culture, the temperature for the scale-up culture was 32 ℃, the culture time was 2.5 days, the temperature for the shaking culture was 32 ℃, the culture time was 2.5 days, and the rotation speed was 150 rpm.
Example 10
This example differs from example 1 only in having a determination15N abundance of N2In the O preparation step, the temperature of shaking culture was 25 ℃, the time was 3.5 days, and the rotation speed was 150 rpm.
Example 11
This example differs from example 1 only in having a determination15N abundance of N2In the O preparation step, the temperature of shaking culture was 32 ℃ for 2.5 days at a rotation speed of 200 rpm.
Example 12
This example differs from example 1 only in that the concentration of the NaOH solution was 1.5 mol/L.
Example 13
This example differs from example 1 only in that the concentration of the NaOH solution was 2.5 mol/L.
Example 14
In the process of nitrogen circulation15NO3 --N and15NO2 -detection of-N and use of the reagent prepared in example 1 with confirmation15N abundance of N2And O is standard gas, and the detection results are calibrated and respectively subjected to 5 parallel tests.
Comparative example 1
By using15N generated by chemical reduction reaction of N-marked nitrite as raw material2And O is standard gas, and the detection results are calibrated and respectively subjected to 5 parallel tests.
The penicillium janthinillum screened by the invention has quick and accurate gas production, and the penicillium janthinillum adopted in the embodiment 1 is calculated according to the dry weight of fungi as follows: yield N per gram dry weight for 7 days of culture2The cumulative amount of O gas is 57.2 g/L; inoculating Penicillium purpureum to liquid Chashi medium according to serum bottle calculation, and measuring N in 1,3,7 days of culture2O release, results are shown in the following table:
| days of culture | Each bottle N2O release amount mg/ |
| 0 | 0 |
| 1 | 34 |
| 3 | 740 |
| 7 | 6858 |
Comparing example 14 with comparative example 1, the deviation between the test result and the true value in example 14 is less than 0.01 μ g and the precision CV is less than 0.7%, while the deviation between the test result and the true value in comparative example 1 is 0.02-0.06 μ g and the precision CV is more than 0.8%
To sum up, the conventional one is avoidedPrepared using chemical methods15N2The problems of inaccurate product content and abundance and difficult storage caused by unstable raw materials in the O process are solved, and the screened N product is creatively used2Penicillium janthinellum with a high O content15N2The preparation of O is simple and quick to operate and low in cost, and the prepared N2O has a definite value15N abundance, having a defined value prepared using the invention15N abundance of N2In the circulation of nitrogen with O as standard gas15NO3 --N and15NO2 -the detection result of the-N is calibrated, the accuracy and precision are obviously improved compared with the standard gas prepared by the traditional oxidation-reduction method, and the accurate conclusion can be obtained in scientific research.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (2)
1. A Penicillium janthinellum strain is preserved in China general microbiological culture Collection center (CGMCC) at 8 months and 7 days in 2017 with the preservation number of CGMCC No. 14146.
2. Based on15Method for measuring nitrogen circulation by N isotope tracer method, characterized in that during nitrogen circulation process15NO3 --N and15NO2 --N, using the Penicillium janthinellum strain of claim 1 for the preparation of a strain with a defined composition15N abundance of N2And O is standard gas to calibrate the detection result.
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| CN102272042A (en) * | 2009-01-08 | 2011-12-07 | 罗伯特·博世有限公司 | Method for obtaining dinitrogen oxide |
| CN102985549A (en) * | 2010-07-07 | 2013-03-20 | 罗伯特·博世有限公司 | Process for obtaining dinitrogen monoxide (N2O) |
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| CN102985549A (en) * | 2010-07-07 | 2013-03-20 | 罗伯特·博世有限公司 | Process for obtaining dinitrogen monoxide (N2O) |
| EP3034600A1 (en) * | 2014-12-16 | 2016-06-22 | Bayer CropScience Biologics GmbH | Method for prolonging the viability of fungal spores in liquid formulations |
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