CN111320158A - An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal - Google Patents

An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal Download PDF

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CN111320158A
CN111320158A CN202010118680.2A CN202010118680A CN111320158A CN 111320158 A CN111320158 A CN 111320158A CN 202010118680 A CN202010118680 A CN 202010118680A CN 111320158 A CN111320158 A CN 111320158A
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卢伟伟
张芳超
查全智
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Nanjing Forestry University
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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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Abstract

The invention discloses an obtaining method13C and15a method for N double-labeling plants and application thereof in preparing biomass charcoal, belonging to the technical field of isotope labeling. The method adopts13C pulse mark and15n foliage spraying method for double marking of plant to obtain13C and15n double-labeled plants and application of double-labeled plants in preparation of13C and15and N double-labeled biomass charcoal. The method is relatively flexible, simple and convenient, has short period of the marking process and high efficiency, is not limited by the growth stage of the plant, is suitable for both pot culture and field in-situ experiments, and gets rid of plant samples13C and15the limitation that N isotope double labeling is difficult to implement and the obtained double-labeled plant is taken as the originalMaterial, preparing13C and15and N double-labeled biomass charcoal.

Description

An obtaining13C and15n double-labeling plant method and application thereof in preparation of biomass charcoal
Technical Field
The invention belongs to the technical field of isotope labeling, and particularly relates to a method for obtaining isotope labeled isotope13C and15a method for N double-marking plants and application thereof in preparing biomass charcoal.
Background
The stable isotope tracing technology can distinguish exogenous substances from soil substances, thereby providing an important technical means for the research of soil element circulation. Biomass char is produced from the pyrolysis of biomass material under oxygen-free or anoxic conditions. Due to the high stability, the biomass charcoal becomes a novel carbon-fixing emission-reducing material. The research on the stability of the biomass charcoal added into the soil and the influence of the biomass charcoal on the element circulation of the soil becomes a research hotspot, and the acquisition of the isotope-labeled biomass charcoal is the basis for the research.
Plants are one of the important raw materials for preparing biomass charcoal. Plant and method for producing the same13C or15Methods of monoisotopic labeling of N are relatively common. However, the plants are simultaneously subjected to13C and15the methods of N isotope double labeling are also relatively limited. The prior art includes: plant stalk using sewing needle and absorbent cotton rope13C and15n double label (CN 104142382A), Syringe injection13CO2Gas and application in silica sand matrix15Obtaining a high-abundance double-labeled plant sample (CN 107047265A) by using the N solution, and introducing the high-abundance double-labeled plant sample by using a high-pressure gas cylinder13CO2Gas and drip irrigation in soil15Device and method for continuously marking plants by using N solution (CN 104221740B), and introducing by using high-pressure gas cylinder13CO2Gas and addition in hydroponic tanks15Continuous application of N solution to aquatic plants13C and15n double-labeled device (CN 207318164U). The existing method is mainly through direct use13CO2Gas is used for plant13C continuous marking, which requires the use of steel cylinders in the implementation, which is inconvenient to transport and presents a certain safety risk. The method for drip irrigation and fertilization is to carry out the steps of15Adding N solution or fertilizer into soil, and allowing plant to absorb through root system15N and assimilated to realize labeling. The nutrient solution and the fertilizer in the two methods need to be added repeatedly, and the time period is long. By sewing needle and absorbent cotton thread15N-urea and13introducing C-glucose solution into plant stalksThe marking method has high recovery rate, but the marking of a large number of plants is not easy to realize. In summary, in order to obtain quickly13C and15n double-labeled biomass charcoal needs to be developed for a method for double labeling plants relatively simply, quickly and efficiently.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for obtaining13C and15the method for marking the plant by N double markers has the advantages of relative flexibility, simplicity, short marking process period, high efficiency, no limitation of the growth stage of the plant, applicability to both pot culture and field in-situ experiments, and getting rid of plant samples13C and15the limitation that double labeling of N isotopes is difficult to implement. The 2 nd technical problem to be solved by the invention is to provide13C and15n double-labeled plants. The 3 rd technical problem to be solved by the invention is to provide13C and15application of N double-labeled plants in preparation of biomass charcoal.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
an obtaining13C and15method for N-double labeling of plants, using13C pulse mark and15n foliage spraying mark to double mark plant to obtain13C and15n double-labeled plants.
Said obtaining13C and15the method for N double-marking plants is carried out in the marking process15Marking of N, then13And C is marked. Such a labeling order is adopted because: (1) if plants are first planted13C mark, the plant can not be immediately de-enzymed, and part of the plant assimilation is realized13C can be utilized by respiration, thereby creating plants13A decrease in C abundance; (2) if plants are first planted13C mark, prolonged standing time of plant, plant13C is transported downward and is concentrated in the roots of the plants and possibly discharged in the form of root exudates, which is disadvantageous for improvement13C, recovering rate; (3)15it still takes a while for the plants to suck after the completion of the N markAbsorbing and assimilating15N, exactly during this time13The C mark can save the marking time and avoid the drawbacks mentioned in (1) and (2) above. Therefore, first-run plants are adopted15Marking of N, then13C, the double-labeled sample contemplated by the present invention can be obtained in a short time. The method specifically comprises the following steps:
(1) removing aged and withered leaves of the plant to be marked, and spraying with atomizing bottle15An N-labeled nitrogen fertilizer solution;
(2) placing the plant in an organic glass box, and under sealed condition, passing through hydrochloric acid and water13C-carbonate reaction production13CO2Pulse marking is carried out, then hydrochloric acid and12c-carbonate reaction production12CO2Each portion of13C-carbonate or12CO production from C-carbonates2The volume concentration of (A) is 300-800 ppm;
(3) and after the marking is finished, taking down the organic glass box, ventilating and finishing the marking process.
Said obtaining13C and15method for N-ditag plants, said15The N-marked nitrogen fertilizer solution is15The concentration of the N-urea solution is 1-3 g/L.
Said obtaining13C and15method for N-ditag plants, said15And adding a surfactant into the N-marked nitrogen fertilizer solution.
Said obtaining13C and15the method for N double-labeling the plant, the surfactant is TritonX-100, and the added volume is15The volume of the N-marked nitrogen fertilizer solution is 0.02-0.08 percent.
Said obtaining13C and15method for N-ditag plants, said13The C-carbonate being Ba13CO3Said12The C-carbonate being Ba12CO3。
Said obtaining13C and15method for N-ditag plants, said13C-carbonate and12fraction of C-carbonateRespectively accounting for 2 parts.
Said obtaining13C and15a method for the N-ditag of a plant which is a herbaceous plant or a dwarf woody plant. The height of the herbaceous or low woody plant should not be greater than the height of the plexiglas box.
Said obtaining13C and15n double-marking plant, wherein the herbaceous plant is rice, wheat, corn or soybean, and the low woody plant is shrub or sapling.
Obtained as described above13C and15n double-labeled plant prepared by method13C and15n double-labeled plants.
As described above13C and15application of N double-labeled plants in preparation of biomass charcoal.
Said13C and15the application of the N double-labeled plant in the preparation of the biomass charcoal comprises the steps of isolating the double-labeled plant from air, and putting the isolated double-labeled plant into a muffle furnace for high-temperature pyrolysis to obtain the biomass charcoal; the temperature rise rate of the muffle furnace is 4-20 ℃/min, and the muffle furnace lasts for 3-6 hours after the target temperature is reached to 250-700 ℃.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention is fast to obtain13C and15the method for N double-marking the plants has the characteristic of strong timeliness; continuous marking is not required, and marking of plants can be completed within 1 day.
(2) The double-marking method of the invention requires simple devices and is easy to operate; does not require storage13CO2The cylinder of gas, no need for an additional power system (such as an electric pump), no need for installation of complicated devices.
(3) The invention is fast to obtain13C and15the method for N double-marking the plant has the characteristic of wide applicability; the method is not limited to pot experiment, and can also be applied to field in-situ experiment; can be applied to various plants, can be herbaceous plants such as rice, wheat, corn, soybean and the like, and can also be short shrubs or saplings; is not limited by the growth stage of the plant.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
Example 1
The plant material used was rice. In 2019, a rice potting experiment was carried out in Shulin farm (119 degrees 13 '28' E, 32 degrees 7 '32' N) under Nanjing forestry university, Zhenjiang, city, Jiangsu province in 5 months. The local climate belongs to subtropical monsoon climate, the annual precipitation is 1055.6mm, the annual average air temperature is 15.2 ℃, and the annual average sunshine hours is 2157 h.
1. Experimental Material
Rice (Oryza sativa L.) with improved resistance to stress13C, marking material: ba13CO3(13C atom 98%, Shanghai chemical research institute), Ba12CO3Hydrochloric acid, a transparent organic glass box, a gas generating device, 704 silicon rubber, a small storage battery fan, an ice box, a thermometer, a hygrometer and black cloth;
(1) transparent organic glass case: to carry out13C, during marking, placing potted rice into an organic glass box (the height of a plant to be marked is not more than the height of the organic glass box), and ensuring that the marking process is carried out under a sealed condition, wherein the box body is made of organic glass with the thickness of about 0.50cm, the length ×, the width × and the height are 106cm, × 71cm, × 100 and 100cm, the box bottom is made of a PVC panel with the thickness of 111cm, × 76cm, the periphery of the panel is provided with a water tank with the height of about 8cm and the width of 4cm, the lower edge of the organic glass box is placed in the water tank, and the box body is sealed by adding water into the water tank;
(2) a gas generating device: the device consists of a 250mL separating funnel, a 250mL beaker and a silicone rubber tube with the outer diameter of 6 mm; mix Ba with13CO3Or Ba12CO3Adding the powder into a beaker, adding hydrochloric acid into a separating funnel, opening the separating funnel, and allowing the hydrochloric acid to enter the beaker through a silicone rubber tube to react with BaCO3Powder reaction to CO2A gas; the gas generating device has 4, 2 generators13CO22 generation of12CO2；
(3)704 silicon rubber: the paint is coated on the organic glass box hole and the joint of the separating funnel to ensure the air tightness;
(4) small battery fan and ice box: the small-sized storage battery fan is hung on the upper bottom surface of the transparent organic glass box and used for uniformly mixing CO generated2A gas; when the temperature in the organic glass box is too high, the temperature is reduced by using an ice box;
(5) thermometer and hygrometer: the temperature and humidity monitoring device is used for monitoring the temperature and the humidity in the organic glass box in real time;
(6) black cloth: CO production in a gas generating apparatus2Front cover organic glass box to promote the generation of CO by rice2The size of the absorption is suitable for just covering the whole organic glass box body.
Rice (Oryza sativa L.) with improved resistance to stress15N-labeled material:
(1)15N-Urea (15N atom 10%, shanghai institute of chemical industry): the concentration of the solution is 0.2% (w/v), namely 2 g/L;
(2) nebulizing bottle (anli corporation): scales are marked, the capacity is 100mL, and the fertilizer is used for foliage spraying;
(3) surfactant (b): TritonX-100, concentration of 0.05% (v/v), surfactant can reduce solution surface tension, inhibit capillary adsorption, slow fertilizer dissolution and recrystallization, increase cell permeability, and promote growth of fertilizer15The N-urea solution is better absorbed.
2. Plant and method for producing the same13C and15n double labeling
The rice is selected to be subjected to isotope labeling at the early reproductive growth stage of rice and in 2019, 9 and 27 months. Firstly, proceed with15Marking of N, followed by13C, the following advantages are achieved by adopting the marking sequence: (1) if plants are first planted13C mark, the plant can not be immediately de-enzymed, and part of the plant assimilation is realized13C can be utilized by respiration, thereby creating plants13A decrease in C abundance; (2) if plants are first planted13C mark, prolonged standing time of plant, plant13C will be transported downwardsThe liquid is transported to and accumulated in the roots of the plants and possibly discharged in the form of root exudates, which is not favorable for improvement13C, recovering rate; (3)15after the N mark is finished, a period of time is still needed for the plant to absorb and assimilate15N, exactly during this time13The C mark can save the marking time and avoid the drawbacks mentioned in (1) and (2) above. The specific operation process is as follows:
(1) removing aged, withered and yellow leaves of potted rice before marking;
(2) using an atomising bottle15Spraying the N solution, adjusting the spray head to fog shape (spray), adjusting the spray amount of the spray head, ensuring that the blades of each rice can be uniformly sprayed, and avoiding excessive spraying to form blade runoff to cause15Waste of N-urea;
(3) after spraying, 3 pots of rice are put into a transparent organic glass box without watering during the period13C, marking; 2 parts of Ba are added before marking13CO3Powder and 2 parts of Ba12CO3The powder was put into 4 beakers of a gas generating apparatus, respectively, each portion of Ba13CO3Or Ba12CO3The mass is 2.65 g;
(4) placing the organic glass box in a base groove, injecting water into the base groove for sealing, and starting the fan; if the temperature in the refrigerator is too high, an ice box can be placed in the refrigerator in advance for cooling;
(5) when marking, the whole organic glass box is covered by black cloth, 200mL of 2mol/L HCl solution and Ba in the 1 st beaker are injected into the separating funnel of the gas generating device13CO3Reaction to produce13CO2Gas, after 5min, opening the black cloth, carrying out photosynthesis on the rice in an organic glass box, and completing the 1 st pulse marking after 45min of photosynthesis; the organic glass box body filled with the rice is covered by black cloth before each marking, so as to carry out hunger treatment and promote the rice to absorb more CO2A gas;
(6) repeating the step (5) to fill Ba in the 2 nd13CO3Adding HCl solution to the beaker to complete the 2 nd time13C, pulse marking;
(7) to improve the rice pair13CO2Repeating the step (5) for 2 times to respectively contain Ba12CO32 beakers add HCl solution to produce12CO2For the absorption and utilization of rice;
(8) and after the marking is finished, taking down the organic glass box, ventilating and harvesting the plants.
3. Isotopic abundance determination
The harvested rice is washed, put into a 105 ℃ oven for deactivation of enzymes for 30min, and then put into a 70 ℃ oven for drying until the weight is constant. Drying, taking out the rice, cooling, sealing and storing. Taking equal amount of rice organ, grinding to particle size of 0.149mm, mixing, and subjecting to stable isotope ratio mass spectrometry (Deltavadvantage, Thermofisher, USA)13C and15the N abundance was measured.
4. Analysis of results
The abundance of the double-labeled rice obtained in this example is shown in Table 1. Overground part and underground part of rice13The difference of C abundance is larger, which indicates that the plant photosynthesis is absorbed in shorter time13C limited transport to the roots. And13c abundance ratio, above ground and underground15The difference in N abundance is small. In conclusion, the method of the invention is successfully adopted to obtain13C and15n double-labeled rice.
TABLE 1 overground and underground part of rice13C and15abundance of N
Figure BDA0002391966850000051
Example 2
2019, 10 and 25, in Shulin farm, Nanjing forestry university, using the method obtained in example 113C and15the method for preparing the biomass charcoal by using the N double-labeled rice comprises the following specific steps:
(1) drying the dried13C and15cutting the N double-marked rice plant into small sections, and crushing by a crusher;
(2) putting the uniformly mixed double-mark rice plants into a crucible, filling and compacting the crucible as much as possible, and ensuring that no gap exists in the crucible;
(3) sealing the crucible by using an aluminum foil to ensure that no crack or broken hole exists on the surface of the aluminum foil so as to ensure that the plants in the crucible are isolated from air;
(4) placing the sealed crucible into a Carbolite muffle furnace (GPC12/131), setting the target temperature to be 300 ℃ and 500 ℃, setting the temperature raising program to be 10 ℃/min, and continuing for 4h after the target temperature is reached;
(5) and closing the muffle furnace, cooling to room temperature, taking out the crucible, and collecting the prepared biomass charcoal.

Claims (13)

1. An obtaining13C and15n double-labeling of plants, characterized in that13C pulse mark and15n method for spraying and marking on leaf surface of plant to obtain double-marked plant13C and15n double-labeled plants.
2. Obtaining according to claim 113C and15n double labeling method for plants, characterized in that the labeling process is performed first15Marking of N, then13And C is marked.
3. Obtaining according to claim 213C and15the method for N double-marking the plant is characterized by comprising the following steps:
(1) removing aged and withered leaves of the plant to be marked, and spraying with atomizing bottle15An N-labeled nitrogen fertilizer solution;
(2) placing the plant in an organic glass box, and under sealed condition, passing through hydrochloric acid and water13C-carbonate reaction production13CO2Pulse marking is carried out, then hydrochloric acid and12c-carbonate reaction production12CO2Each portion of13C-carbonate or12CO production from C-carbonates2The volume concentration of (A) is 300-800 ppm;
(3) and after the marking is finished, taking down the organic glass box, ventilating and finishing the marking process.
4. Obtaining according to claim 313C and15method for N-double labeling a plant, characterized in that15The N-marked nitrogen fertilizer solution is15The concentration of the N-urea solution is 1-3 g/L.
5. Obtaining according to claim 3 or 413C and15method for N-double labeling a plant, characterized in that15And adding a surfactant into the N-marked nitrogen fertilizer solution.
6. Obtaining according to claim 513C and15the method for N double-marking the plants is characterized in that the surfactant is TritonX-100, and the added volume is15The volume of the N-marked nitrogen fertilizer solution is 0.02-0.08 percent.
7. Obtaining according to claim 313C and15method for N-double labeling a plant, characterized in that13The C-carbonate being Ba13CO3Said12The C-carbonate being Ba12CO3。
8. Obtaining according to claim 3 or 713C and15method for N-double labeling a plant, characterized in that13C-carbonate and12the parts of the C-carbonate are respectively 2 parts.
9. Obtaining according to claim 113C and15a method of N-double labelling a plant, wherein said plant is a herbaceous plant or a dwarf woody plant.
10. Obtaining according to claim 913C and15the method for N double-marking plants is characterized in that the herbaceous plants are rice, wheat, corn or soybean; what is needed isThe low woody plant is shrub or sapling.
11. The method of claim 113C and15n double-labeled plant prepared by method13C and15n double-labeled plants.
12. The method of claim 1113C and15application of N double-labeled plants in preparation of biomass charcoal.
13. The method of claim 1213C and15the application of the N double-labeled plant in the preparation of the biomass charcoal is characterized in that the double-labeled plant is isolated from air and then put into a muffle furnace for high-temperature pyrolysis to obtain the biomass charcoal; the temperature rise rate of the muffle furnace is 4-20 ℃/min, and the muffle furnace lasts for 3-6 hours after the target temperature is reached to 250-700 ℃.
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