WO2021169154A1 - Method for obtaining 13c and 15n dual-labeled plant and use thereof in preparation of biomass charcoal - Google Patents

Abstract

Disclosed are a method for obtaining a ¹³C and ¹⁵N dual-labeled plant and the use thereof in the preparation of biomass charcoal. The present invention belongs to the technical field of isotope labeling. The method involves dual-labeling a plant using a method of ¹³C pulse labeling and ¹⁵N foliage spraying to obtain a ¹³C and ¹⁵N dual-labeled plant. The dual-labeled plant is used for preparing ¹³C and ¹⁵N dual-labeled biomass charcoal. The method of the present invention is relatively flexible, simple and convenient, has a short period for the labeling process and a high efficiency, is not limited by the growth stage of the plant, is applicable to both potting and in situ field experiments, and gets rid of the limitation that the dual-labeling of a plant sample with ¹³C and ¹⁵N isotopes is hard to implement. In addition, ¹³C and ¹⁵N dual-labeled biomass charcoal is prepared from the obtained dual-labeled plant as a raw material.

Description

A gain 13C and 15N double-labeled plant method and its application in preparing biomass charcoal Technical field

The invention belongs to the technical field of isotope labeling, and specifically relates to a method for obtaining ¹³ C and ¹⁵ N double-labeled plants and its application in the preparation of biomass charcoal.

Background technique

Stable isotope tracing technology can distinguish between exogenous materials and soil itself, thereby providing important technical means for the study of soil element cycle. Biomass charcoal is produced by the pyrolysis of biomass materials under anaerobic or anaerobic conditions. Due to its high stability, biomass charcoal has become a new type of carbon sequestration and emission reduction material. Research on the stability of biomass charcoal added to the soil and its influence on the element cycle of the soil itself has become a research hotspot, and obtaining isotope-labeled biomass charcoal is the basis for this research.

Plants are one of the important raw materials for the preparation of biomass charcoal. The method of monoisotopic labeling of plants with ¹³ C or ^{15 N is relatively common.} However, ^{the methods for dual-labeling plants with 13} C and ¹⁵ N isotopes are still relatively limited. Existing published technologies include: using sewing needles and absorbent cotton ropes for ¹³ C and ¹⁵ N double marking of plant stems (CN 104142382 A), syringe injection of ¹³ CO ₂ ^{gas and application of 15} N solution in quartz sand matrix to obtain high abundance Dual-labeled plant samples (CN 107047265 A), using a high-pressure gas cylinder to pass ¹³ CO ₂ gas and drip irrigation ¹⁵ N solution in the soil to continuously mark plants and methods (CN 104221740 B), using a high-pressure gas bottle to pass ¹³ CO ₂ A device (CN 207318164 U) ^{for continuous 13} C and ¹⁵ N double labeling of aquatic plants by ^{adding 15} N solution to gas and a hydroponic tank. The existing method mainly uses ¹³ CO ₂ gas to perform ¹³ C continuous labeling on plants, which requires the use of steel cylinders during the implementation process, which is inconvenient to transport and poses a certain safety risk. The method of drip irrigation and fertilization is to add ¹⁵ N solution or fertilizer to the soil and other substrates. The plant absorbs ¹⁵ N through roots and assimilates to achieve marking. The nutrient solution and fertilizer in the two methods need to be added repeatedly, and the time period is long. ^{The labeling method in which 15} N-urea and ¹³ C-glucose solutions are introduced into plant stems through sewing needles and absorbent cotton thread has a high recovery rate, but it is not easy to mark a large number of plants. In summary, in order to quickly obtain ¹³ C and ¹⁵ N dual-labeled biomass char, it is necessary to first develop a relatively simple, fast and efficient method for dual-labeling plants.

Summary of the invention

The technical problem to be solved by the present invention is to provide a method for obtaining ¹³ C and ¹⁵ N double-labeled plants. The labeling method is relatively flexible and simple, the labeling process has a short cycle and high efficiency, and is not limited by the growth stage of plants. In situ experiments are all applicable, and get rid of the limitation that the dual labeling of ¹³ C and ^{15 N isotopes is difficult to implement in plant samples.} The second technical problem to be solved by the present invention is to provide a ¹³ C and ¹⁵ N double-labeled plant. The third technical problem to be solved by the present invention is to provide an application of ¹³ C and ¹⁵ N double-labeled plants in the preparation of biomass charcoal.

In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

A method for obtaining ¹³ C and ¹⁵ N double-labeled plants, adopts ¹³ C pulse labeling and ¹⁵ N foliar spray labeling to double-label the plants to obtain ¹³ C and ¹⁵ N double-labeled plants.

In the method for obtaining ¹³ C and ¹⁵ N double-labeled plants, ¹⁵ N labeling is performed ^{first, and then 13} C labeling is performed during the labeling process. This labeling sequence is adopted because: (1) If the ¹³ C labeling of plants is carried out ^{first, the plants cannot be killed in time, and a part of the 13} C assimilation of plants can be used through respiration, resulting in a decrease in the abundance of ^{13 C in plants;} (2) If the plant ¹³ C labeling is performed first, the plant's standing time is prolonged, the plant ¹³ C will be transported downwards, and it will be enriched in the roots of the plant and may be excreted in the form of root exudates, which is not conducive to increasing the ¹³ C. Recovery rate: (3) After ¹⁵ N labeling is completed, it still takes a while for plants to absorb and assimilate ¹⁵ N. It happens that ¹³ C labeling during this period can save labeling time and avoid the above (1) and (2). To the drawbacks. Therefore, the ¹⁵ N labeling of ^{plants and then the 13} C labeling method can be adopted to obtain the double-labeled sample expected by the present invention in a short time. It includes the following steps:

(1) Remove the senescent and yellow leaves of the plants to be marked, and spray a ¹⁵ N-labeled nitrogen fertilizer solution with an atomized bottle;

(2) Put the plant in a plexiglass box, under airtight conditions, firstly ^{generate 13} CO ₂ ^{by the reaction of hydrochloric acid and 13} C-carbonate for pulse labeling, and then ^{generate 12} CO _{2 by the} ^{reaction of hydrochloric acid and 12} C-carbonate , _{The volume concentration of CO 2} ^{produced by each 13} C-carbonate or ¹² C-carbonate is 300～800ppm;

(3) After marking, remove the plexiglass box, ventilate, and complete the marking process.

In the method for obtaining ¹³ C and ¹⁵ N dual-labeled plants, the ¹⁵ N-labeled nitrogen fertilizer solution is a ¹⁵ N-urea solution with a concentration of 1 to 3 g/L.

In the method for obtaining ¹³ C and ¹⁵ N double-labeled plants, a ^{surfactant is added to the 15} N-labeled nitrogen fertilizer solution.

In the method for obtaining ¹³ C and ¹⁵ N double-labeled plants, the surfactant is Triton X-100, and the volume of the ¹⁵ N-labeled nitrogen fertilizer solution is 0.02% to 0.08%.

In the method for obtaining ¹³ C and ¹⁵ N dual-labeled plants, the ¹³ C-carbonate is Ba ¹³ CO ₃ and the ¹² C-carbonate is Ba ¹² CO ₃ .

In the method for obtaining ¹³ C and ¹⁵ N dual-labeled plants, the number of ¹³ C-carbonate and ¹² C-carbonate is 2 parts respectively.

In the method for obtaining ¹³ C and ¹⁵ N double-labeled plants, the plants are herbaceous plants or low woody plants. The height of herbaceous plants or low woody plants should not be greater than the height of the plexiglass box.

In the method for obtaining ¹³ C and ¹⁵ N double-labeled plants, the herbaceous plants are rice, wheat, corn or soybeans, and the low woody plants are shrubs or saplings.

¹³ C and ¹⁵ N double-labeled plants prepared by the above method for obtaining ¹³ C and ¹⁵ N double-labeled plants.

Application of the above-mentioned ¹³ C and ¹⁵ N double-labeled plants in the preparation of biomass charcoal.

The application of the ¹³ C and ¹⁵ N double-labeled plants in the preparation of biomass charcoal, the double-labeled plants are isolated from air and put into a muffle furnace for high-temperature pyrolysis to obtain biomass charcoal; the heating rate of the muffle furnace It is 4～20℃/min, and lasts for 3～6h after reaching the target temperature of 250～700℃.

Compared with the existing technology, the beneficial effects of the present invention include:

(1) The method of the present invention for quickly obtaining ¹³ C and ¹⁵ N double-labeled plants has the characteristics of strong timeliness; continuous labeling is not required, and the labeling of plants can be completed within one day.

(2) The device required for the dual marking method of the present invention is simple and easy to operate; it does not require ^{a steel cylinder for storing 13} CO ₂ gas, does not require an additional power system (such as an electric pump), and does not require the installation of complex devices.

(3) The method for quickly obtaining ¹³ C and ¹⁵ N double-labeled plants of the present invention has the characteristics of wide applicability; it is not limited to pot experiments, but can also be applied to in-situ experiments in the field; it can be applied to a variety of plants, including rice Herbs such as wheat, corn, soybeans, etc., can also be low shrubs or saplings; they are not limited by the growth stage of the plant.

Detailed ways

In order to make the above objectives, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention will be described in detail below in conjunction with specific embodiments.

Example 1

The plant material used is rice. In May 2019, the rice pot experiment was carried out at Xiashu Forest Farm (119°13′28″E, 32°7′32″N) of Nanjing Forestry University, Zhenjiang City, Jiangsu Province. The local climate belongs to the subtropical monsoon climate, the annual precipitation is 1055.6mm, the annual average temperature is 15.2℃, and the annual average sunshine hours is 2157h.

1. Experimental materials

Rice ¹³ C marking materials: Ba ¹³ CO ₃ ( ¹³ C atom 98%, Shanghai Research Institute of Chemical Industry), Ba ¹² CO ₃ , hydrochloric acid, transparent plexiglass box, gas generator, 704 silicone rubber, small battery fan, ice box, Thermometer and hygrometer, black cloth;

(1) Transparent plexiglass box: when performing ¹³ C marking, put potted rice in the plexiglass box (the height of the plant to be marked should not be greater than the height of the plexiglass box) to ensure that the marking process is carried out in a sealed condition. The box body is made of plexiglass with a thickness of about 0.50cm, length×width×height is 106cm×71cm×100cm, and the bottom of the box is made of a 111cm×76cm PVC panel. There is a sink with a height of about 8cm and a width of 4cm around the panel. The bottom edge of the plexiglass box is placed in the sink, and the box is sealed by adding water to the sink; four holes with a diameter of about 2.30cm are evenly designed on the top of the plexiglass box to install the separatory funnel; one on each side of the box Handle to facilitate the movement of the transparent plexiglass box;

(2) Gas generating device: consists of a 250mL separatory funnel, a 250mL beaker and a silicone rubber tube with an outer diameter of 6mm; add Ba ¹³ CO ₃ or Ba ¹² CO ₃ powder into the beaker, and add hydrochloric acid to the separatory funnel, After opening the separatory funnel, the hydrochloric acid enters the beaker through the silicone rubber tube and _{reacts with the BaCO 3} powder to generate CO ₂ gas; there are 4 gas generators, 2 generating ¹³ CO ₂ , and 2 generating ¹² CO ₂ ;

(3) 704 silicone rubber: apply to the plexiglass box holes and the joints of the separatory funnel to ensure air tightness;

(4) Small battery fan and ice box: The small battery fan is suspended on the upper and bottom surface of the transparent plexiglass box to mix the CO ₂ gas generated; when the temperature in the plexiglass box is too high, use an ice box to cool it down;

(5) Thermometer and hygrometer: used to monitor the temperature and humidity in the plexiglass box in real time;

(6) Black cloth: Cover the plexiglass box before the _{CO 2 is produced by the gas generator to promote the absorption of the CO 2} produced by the rice. The size should just cover the entire plexiglass box.

Rice ¹⁵ N-labeled materials:

(1) ¹⁵ N-urea ( ¹⁵ N atom 10%, Shanghai Research Institute of Chemical Industry): the solution concentration is 0.2% (w/v), which is 2g/L;

(2) Atomization bottle (Amway Company): marked with a scale, with a capacity of 100mL, used for foliar spray;

(3) Surfactant: Triton X-100, with a concentration of 0.05% (v/v). Surfactant can reduce the surface tension of the solution, inhibit capillary adsorption, and slow down the dissolution and recrystallization process of fertilizers. Cell permeability promotes ^{better absorption of 15} N-urea solution.

2. Plant ¹³ C and ¹⁵ N double labeling

The early stage of reproductive growth of rice was selected, and the isotope labeling of rice was carried out on September 27, 2019. First carry out the ¹⁵ N labeling, and then carry out the ¹³ C labeling. Using this labeling sequence has the following advantages: (1) If the plant ¹³ C labeling is carried out first, the plant cannot be killed in time, and a part of the ¹³ C assimilation of the plant can pass Respiration is used to ^{reduce the 13} C abundance of plants; (2) If the plant ¹³ C labeling is carried out first, the plant’s standing time ^{will be prolonged, and the plant 13} C will be transported downwards and accumulated in the roots of the plant. It may be excreted in the form of root exudates, which is not conducive to improving the ¹³ C recovery rate; (3) After the ¹⁵ N labeling is completed, it still takes a while for the plant to absorb and assimilate ¹⁵ N. It happens that the ¹³ C labeling during this period can save labeling. Time, and will avoid the drawbacks mentioned in (1) and (2) above. The specific operation process is as follows:

(1) Remove the senescent and yellow leaves of potted rice before marking;

(2) Use an atomizing bottle to spray the ¹⁵ N solution, adjust the nozzle to spray, adjust the spray amount of the nozzle to ensure that the leaves of each rice plant can be sprayed evenly, and avoid excessive spraying to form leaf runoff. Cause ^{a waste of 15} N-urea;

(3) After spraying, do not water during the period. Put 3 pots of rice in a transparent plexiglass box and ^{mark with 13} C; before marking, put 2 parts of Ba ¹³ CO ₃ powder and 2 parts of Ba ¹² CO ₃ powder separately Into the 4 beakers of the gas generating device, the ^{mass of each portion of Ba 13} CO ₃ or Ba ¹² CO ₃ is 2.65g;

(4) Put the plexiglass box in the base trough, pour water into the base trough to seal, and start the fan; if the temperature in the box is too high, you can put an ice box in the box to cool down in advance;

(5) When marking, first cover the entire plexiglass box with a black cloth, and pour 200mL 2mol/L HCl solution into the separatory funnel of the gas generator, and react with the Ba ¹³ CO ₃ in the first beaker to produce ¹³ CO ₂ gas After 5 minutes, the black cloth was opened, the rice was photosynthesized in the plexiglass box, and the first pulse marking was completed after 45 minutes of photosynthesis; the plexiglass box containing the rice was covered with a black cloth before each marking, yes In order to deal with starvation, encourage rice to absorb more CO ₂ gas;

(6) Repeat step (5), ^{add HCl solution to the second beaker containing Ba 13} CO ₃ to complete the second ¹³ C pulse mark;

(7) In order to improve ^{the absorption of 13} CO ₂ by rice, repeat step (5) twice, and add HCl solution to the two beakers ^{containing Ba 12} CO ₃ ^{to produce 12} CO ₂ for rice absorption and utilization;

(8) After the marking is over, remove the plexiglass box, ventilate, and harvest the plants.

3. Determination of isotope abundance

After washing the harvested rice, put it in an oven at 105°C for 30 minutes, and then put it in an oven at 70°C to bake to constant weight. After drying, the rice is taken out, cooled, and sealed for storage. Take an equal amount of samples from each organ of rice, fully grind them to a particle size of 0.149 mm, mix them, and use a stable isotope ratio mass spectrometer (DELTA V Advantage, Thermofisher, USA) to determine the ¹³ C and ¹⁵ N abundance of the rice samples. Degree is measured.

4. Result analysis

The abundance of the double-labeled rice obtained in this example is shown in Table 1. There is a large difference in the abundance of ¹³ C between the aboveground and underground parts of rice ^{, which indicates that the 13} C absorbed by photosynthesis of plants is limited in transportation to the roots in a relatively short period of time. Compared with ¹³ C abundance, the difference in ¹⁵ N abundance above ground and underground is smaller. In summary, the method of the present invention has successfully obtained ¹³ C and ¹⁵ N double-labeled rice.

^{Table 1 13} C and ¹⁵ N abundances in the aboveground and underground parts of rice

Example 2

On October 25, 2019, in the Xiashu Forest Farm of Nanjing Forestry University, the ¹³ C and ¹⁵ N double-labeled rice obtained in Example 1 was used to prepare biomass char, and the ^{abundance of 13} C and ¹⁵ N of the biomass char was measured.

1. The preparation process of biomass charcoal

(1) Cut the dried ¹³ C and ¹⁵ N double-labeled rice plants into small sections and crush them with a pulverizer;

(2) Put the mixed double-labeled rice plants into the crucible, fill the crucible as much as possible, and ensure that there are no voids inside;

(3) Seal the crucible with aluminum foil to ensure that there are no cracks or holes on the surface of the aluminum foil to ensure that the plants in the crucible are isolated from air;

(4) Put the sealed crucible into the Carbolite muffle furnace (GPC12/131), set the target temperature to 300°C and 500°C, set the heating program to 10°C/min, and continue for 4 hours after reaching the target temperature;

(5) Turn off the muffle furnace, cool to room temperature, take out the crucible, and collect the prepared biomass charcoal.

^{2. Determination of 13} C and ¹⁵ N abundance of biomass charcoal

^{The 13} C and ¹⁵ N abundances of the prepared biomass charcoal are shown in Table 2. Compared with plant raw materials, the ¹³ C abundance of biomass charcoal prepared from rice decreased, especially in the underground parts of rice, which decreased by 69.2% and 71.3% at 300°C and 500°C, respectively. ^The abundance of 15 N also decreased, but the degree of decrease was lower than that of ¹³ C. At 500°C, compared with plant raw materials, the ^{15 N abundance of biomass charcoal prepared from the aboveground part of rice hardly changed, while the 15} N abundance of biomass charcoal prepared from the underground part decreased by 40.5%.

^{Table 2 13} C and ¹⁵ N abundances of biomass char prepared from aboveground and underground parts of rice

Claims (13)

1. A method for obtaining ¹³ C and ¹⁵ N double-labeled plants is characterized in that the ¹³ C pulse labeling and ¹⁵ N foliar spraying labeling methods are used to double-label the plants to obtain ¹³ C and ¹⁵ N double-labeled plants.
2. ^{The method for obtaining 13} C and ¹⁵ N double-labeled plants according to claim 1, characterized in that, during the labeling process, ¹⁵ N labeling is performed ^{first, and then 13} C labeling is performed.
3. ^{The method for obtaining 13} C and ¹⁵ N double-labeled plants according to claim 2, characterized in that it specifically comprises the following steps:

   (1) Remove the senescent and yellow leaves of the plants to be marked, and spray a ¹⁵ N-labeled nitrogen fertilizer solution with an atomized bottle;

   (2) Put the plant in a plexiglass box, under airtight conditions, firstly ^{generate 13} CO ₂ ^{by the reaction of hydrochloric acid and 13} C-carbonate for pulse labeling, and then ^{generate 12} CO _{2 by the} ^{reaction of hydrochloric acid and 12} C-carbonate , _{The volume concentration of CO 2} ^{produced by each 13} C-carbonate or ¹² C-carbonate is 300～800ppm;

   (3) After marking, remove the plexiglass box, ventilate, and complete the marking process.
4. ^{The method for obtaining 13} C and ¹⁵ N dual-labeled plants according to claim 3 ^{, wherein the 15} N-labeled nitrogen fertilizer solution is a ¹⁵ N-urea solution with a concentration of 1 to 3 g/L.
5. ^{The method for obtaining 13} C and ¹⁵ N dual-labeled plants according to claim 3 or 4, wherein a ^{surfactant is added to the 15} N-labeled nitrogen fertilizer solution.
6. ^{The method for obtaining 13} C and ¹⁵ N dual-labeled plants according to claim 5, wherein the surfactant is Triton X-100, and the added volume is ^{0.02 of the volume of the 15} N-labeled nitrogen fertilizer solution. %～0.08%.
7. ^{The method for obtaining 13} C and ¹⁵ N dual-labeled plants according to claim 3 ^{, wherein the 13} C-carbonate is Ba ¹³ CO ₃ and the ¹² C-carbonate is Ba ¹² CO ₃ .
8. ^{The method for obtaining 13} C and ¹⁵ N dual-labeled plants according to claim 3 or 7, characterized in that the parts of ¹³ C-carbonate and ¹² C-carbonate are 2 parts respectively.
9. ^{The method for obtaining 13} C and ¹⁵ N double-labeled plants according to claim 1, wherein the plants are herbaceous plants or low woody plants.
10. ^{The method for obtaining 13} C and ¹⁵ N double-labeled plants according to claim 9, wherein the herbaceous plant is rice, wheat, corn or soybean; and the low woody plant is a shrub or sapling.
11. Preparation method for obtaining ¹³ C and ¹⁵ N double-labeled plant obtained ¹³ C and ¹⁵ N double-labeled plant according to claim 1.
12. The application of the ¹³ C and ¹⁵ N dual-labeled plants as claimed in claim 11 in the preparation of biomass charcoal.
13. ^{The application of 13} C and ¹⁵ N dual-labeled plants in the preparation of biomass charcoal according to claim 12, characterized in that the dual-labeled plants are isolated from air and placed in a muffle furnace for high-temperature pyrolysis to obtain biomass char; The heating rate of the muffle furnace is 4-20°C/min, and it lasts for 3-6 hours after reaching the target temperature of 250-700°C.