CN114002361B - Dry land soil background N 2 Method for measuring discharge rate - Google Patents
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Abstract
The invention provides a dry land soil background N 2 Methods for determining discharge rate, includingThe following steps: step 1, adding 30g of air-dried soil into a 120ml serum bottle; step 2, placing the rubber spacer in a He environment in advance; step 3, regulating the water content of the soil sample in the serum bottle to be 30% of the mass water content by using distilled water filled with He for treatment for 3 hours; step 4, sealing the serum bottle by using a rubber spacer which is placed in the He environment in advance and compacting by using an aluminum cover; step 5, replacing headspace gas in the serum bottle by adopting a He flushing mode; step 6, balancing the gas pressure in the serum bottle by using a 10ml piston-free injector containing distilled water; step 7, placing the serum bottle into an anaerobic box with He as a gas source to continue culturing for 48 hours; and 8, quantifying the gas in the serum bottle by adopting a destructive sampling mode. The invention is realized by pairing N possibly generated 2 Each link of leakage is optimized, and the external N is reduced to the maximum extent 2 The leakage of the soil is realized, and the dry land soil background N is realized 2 Accurate determination of discharge rate.
Description
Technical Field
The invention belongs to the technical field of denitrification determination, and particularly relates to a dry land soil background N 2 Method for measuring discharge rate.
Background
Worldwide, active nitrogen produced by human activities shows a tendency to rise year by year. Excessive amounts of active nitrogen enter the environment and pose a serious threat to the quality of the ecological environment. Denitrification is the conversion of active nitrogen to inert nitrogen (N) in the environment 2 ) However due to the presence of higher concentrations of background N in the atmosphere 2 (78%), making it difficult to accurately quantify N produced by the ecosystem denitrification process 2 Flux.
At present, for river, lake sediment and rice field ecosystems, as the flooding interface can realize a certain degree of retardation effect on gas exchange, the nitrogen gas can pass through N 2 Method for realizing generation of N in denitrification process by adopting/Ar combined membrane sample introduction mass spectrometry 2 Accurate quantification of (3). However, for dry land soils, direct andaccurate determination of N 2 The discharge rate remains a significant challenge.
In recent years, researchers developed a direct denitrifying gas measurement system, namely a Robot system (Robotized accumulation and analysis system), which can realize the N-phase culture system 2 Direct measurement of (2). The principle is that the low background N is artificially created by the He flushing mode 2 The culture environment of the culture system, and further realizes the generation of trace N in the denitrification process in the culture system 2 Direct quantization of (2). However, the prior art is more suitable for N in a denitrifying bacteria pure bacteria culture system 2 Determination of the discharge Rate while the pure bacteria System denitrifies N under appropriate conditions 2 The discharge rate is higher, and N with higher concentration can be generated in shorter time 2 Even if a slight amount of N is present in the system 2 Leakage does not significantly affect the results of the assay, and therefore is not detrimental to the external N 2 The control steps of leakage are less, and the system has inevitable N in the culture and measurement process 2 Leakage occurs. But due to the high concentration of N in the environment 2 Background (78%) once N in serum bottle 2 The discharge rate is low even if the serum bottle has trace N 2 Leakage will also seriously affect the assay result, and more accurate N cannot be obtained by using the method for determination 2 The rate of discharge. While for dry land soil, its background without fertilization is N 2 The discharge rate is relatively low and is easily subjected to the trace N generated by the system 2 The influence of leakage causes that the original method cannot meet the analysis and test requirements, and a new method is urgently needed to realize the dry land soil background N 2 And (4) measuring the discharge rate.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a dry land soil background N 2 The method for measuring the discharge rate of the present invention is carried out by measuring the production of N during the culturing and measuring process 2 Optimized control of each link of leakage, and maximally reducing outside N 2 The leakage of the soil can further realize the dry land soil N 2 Accurate determination of discharge rate.
The invention adopts the following technical scheme:
dry land soil background N 2 A method of determining a discharge rate comprising the steps of:
step 1, adding 30g of air-dried soil into a 120ml serum bottle;
step 2, treating the rubber spacer by using a He (purity of 99.999%) flushing mode to pre-place the rubber spacer in a He environment;
step 3, regulating the water content of the soil sample in the serum bottle by using distilled water to ensure that the water content of the soil sample is 30 mass percent;
step 4, coating a small amount of vacuum lubricating silicone grease on the opening of the serum bottle, sealing the opening by using a rubber spacer which is placed in the He environment in advance, and compacting by using an aluminum cover;
step 5, replacing headspace gas in the serum bottle by using a He flushing mode;
the method specifically comprises the steps of vacuumizing a serum bottle for 300s, filling the serum bottle with high-purity He (with the purity of 99.999%) for 30s, and repeating the steps for 6 times, wherein the filling pressure is 1.5 MPa;
step 7, placing the serum bottle into an anaerobic tank to reduce the external N 2 Coating a layer of 704 sealing silica gel outside the rubber spacer to further prevent the leakage of outside air, and continuously culturing for 48h;
step 8. The headspace gas concentration in the serum vial was determined at regular intervals using gas chromatography (7890b, agilent, usa).
The further technical scheme is that the concrete steps of flushing the rubber spacer by He in the step 2 comprise:
(1) To a 120ml volume cylinder (svg 100, nichiden-rika, japan) was placed 20 rubber spacers and sealed and then the cylinder was He-rinsed;
the method specifically comprises the steps of vacuumizing a cylindrical bottle for 300s, filling high-purity He (purity is 99.999%) for 30s under the filling pressure of 1.5MPa, and repeating for 6 times;
(2) And placing for 24h, then performing He flushing treatment again, and continuing to place for 24h for later use.
The further technical scheme is that the distilled water used for adjusting the water content of the soil in the step 3 needs to be filled with He (with the purity of 99.999%) in advance for treatment for 3 hours.
The further technical scheme is that 2ml of distilled water is contained in the 10ml of piston-free syringe in the step 6, and when the syringe is inserted into the serum bottle, air can be prevented from entering the serum bottle, so that the effect of sealing and isolating air exchange is achieved.
The further technical proposal is that in the step 7, the anaerobic tank uses He (with the purity of 99.999%) as a gas source, and low N is built inside the tank body 2 And (4) environment.
The further technical scheme is that step 8 specifically comprises sampling in a destructive sampling mode, that is, more parallels are set for each group of treatment, and all serum bottles are only sampled and measured once.
The invention has the beneficial effects that:
the method of the invention can generate N due to strengthening 2 The leakage control of each leakage link can greatly reduce the leakage rate of the sample during the culture period, thereby realizing the trace N generated by the culture system 2 The accuracy of the measurement.
Drawings
FIGS. 1 (a) -1 (b) are flow charts of the steps of the present invention;
FIG. 2 is a graph showing the leakage rate measured using the background art;
FIG. 3 shows the leakage rate measured by the method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described below clearly and completely, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention replaces the original culture substrate bacteria liquid with the dry land soil sample, but the N of the dry land soil 2 Lower discharge rate if the prior art is used (background)Methods in the art) will generate large errors and thus to achieve a dry land soil background N 2 Accurate quantification of discharge rate, the present invention by comparing the potential generation of N 2 Optimizing each link of leakage to reduce outside N to the maximum extent 2 Thereby realizing a trace of N 2 The accurate determination of (2).
As shown in fig. 1 (b), (1) step: preparing a sample to be tested, (2) steps: preparing a cylindrical bottle for He washing of the rubber spacer (the gray square in the bottle is the rubber spacer), (3): adjusting the water content process by adding He-filled water into the soil sample in the serum bottle, (4) steps: sealing the serum bottle by using a rubber spacer which is placed in the He environment in advance, (5) step: replacing the headspace of the serum bottle by He flushing, (6) step: balancing the serum bottle pressure using a 10ml syringe containing 2ml of distilled water, (7) steps: culturing in an anaerobic box (the state shown in the figure is that the sample to be tested is just pre-cultured in the anaerobic box), (8): measured using a gas chromatograph.
Note: the He of the invention does not mark the purity, and the purity of the He is 99.999 percent.
As shown in FIGS. 1 (a) to 1 (b), a dry land soil background N of the present invention 2 A method of determining a discharge rate comprising the steps of:
step 1, 30g of air-drying soil is added into a serum bottle.
Step 2, processing the rubber spacer by using a He (purity of 99.999%) flushing mode to enable the rubber spacer to be placed in a He environment in advance, wherein the He flushing rubber spacer comprises the following specific steps: 20 rubber spacers were put into a 120ml cylindrical bottle (svg 100, nichiden-rika, japan) and sealed, and then the cylindrical bottle was subjected to He rinsing (vacuum treatment 300s was performed on the cylindrical bottle, then high purity He, purity 99.999% was used, 30s was filled, filling pressure 1.5MPa was repeated 6 times) for 24 hours, and then He rinsing treatment was performed again and continued for 24 hours for backup.
Step 3, adjusting the water content of the soil to be 30% by mass (according to the collected dry land soil samples, the mass water content of most samples of the soil is found to be about 20%, so that the water content is set to be 30%, and the water among the samples is unified while the actual conditions in the field are restored as much as possibleFractional content), distilled water for adjusting the water content of soil needs to be pre-filled with He for 3 hours because a certain concentration of N is dissolved in water at room temperature 2 When distilled water is added to the flask, external N is introduced into the culture system 2 Therefore, to minimize N 2 The contamination of the atmosphere is minimized by the treatment with He (purity 99.999%).
And 4, coating a small amount of vacuum lubricating silicone grease on the opening of the serum bottle, sealing the serum bottle by using a rubber spacer in a He (purity of 99.999%) environment, and compacting by using an aluminum cover.
This step was carried out because it was found in experiments that a certain amount of N was adsorbed in the rubber spacers 2 The N absorbed in the serum bottle after the serum bottle is sealed by using a rubber spacer which is not subjected to any treatment 2 Can exchange with the headspace gas in the serum bottle, especially after the serum bottle is subjected to He flushing process, because the headspace gas of the serum bottle is low N 2 The environment, the diffusion function is particularly prominent, and N is greatly influenced 2 And (4) measuring the discharge rate. The rubber spacer pre-placed in the He environment can complete part of gas exchange process in the He environment, so that the serum bottle is sealed by using the rubber spacer pre-placed in the He environment, and N contained in the rubber spacer can be controlled to a certain extent 2 Exchange gas with the headspace of the serum bottle.
And 5, replacing headspace gas in the serum bottle by using a He (purity of 99.999%) flushing mode, wherein the specific process comprises the steps of vacuumizing the serum bottle for 300s, filling the serum bottle with high-purity He (purity of 99.999%) for 30s, and repeatedly performing 6 times at the filling pressure of 1.5 MPa.
Step 7, placing the serum bottle into an anaerobic box (the anaerobic box uses He (purity is 99.999%) as a gas source, and creating low N in the box 2 Environment) reducing the outside N 2 And a layer 704 of sealing silica gel is coated on the rubber spacer to further prevent the leakage of outside air. And continuously culturing in an anaerobic box for 48 hours. This step is carried outThe reason for the cultivation is that not only a certain concentration of N is dissolved in water 2 N with a certain concentration is also adsorbed in soil pores of the soil sample in the bottle 2 N from both sources 2 Gradually diffusing out the He after the serum bottle finishes He flushing through the exchange effect with headspace gas in the serum bottle, and reaching balance within a certain time so as to further eliminate N from the two sources 2 Leakage, the incubation treatment of this step is required.
Step 8. Headspace gas was measured at regular intervals using gas chromatography (7890b, agilent, usa). The sampling is carried out in a destructive sampling mode, namely more parallels are arranged for each group of treatment, and all serum bottles are sampled and measured once (because the rubber spacer blocks N in the air) 2 The only medium diffused into the serum bottle can generate larger wound by puncture sampling for a plurality of times, so that the leakage quantity is increased continuously, and the wound is reduced as much as possible by adopting a destructive sampling mode so as to reduce N 2 The purpose of the leak).
Examples
In this example, the technical solution proposed in the present invention is used to demonstrate N in the determination of dry land soil samples 2 The specific implementation effect of the leakage condition control is as follows:
collecting 0-20 cm surface soil under a facility vegetable planting system near a Chinese academy of sciences perennial agriculture ecological experiment station, sealing the collected soil by using a sealing bag, conveying the soil to a laboratory as soon as possible, air-drying the soil, uniformly sieving the soil by using a 2mm sieve, and removing impurities for later use.
1) Earth pillar sample preparation
After the air-dried soil sample passing through the 2mm sieve was sterilized with high-pressure steam for 2 hours, 30g of the soil sample was weighed and placed in a 120ml serum bottle. A total of two treatments were designed in the experiment. The following were used 1) and 2) for the measurement using the method proposed by the present invention, respectively. The soil sample used in the experiment is sterilized, and N generated by soil abiotic process 2 The discharge amount can be ignored, so the measured N in the experiment 2 The discharge rate is N 2 The leak rate of (c).
2) Specific assay procedures for different treatments
For the processing of the measurement using the background art, the specific measurement procedures are as follows: adding untreated distilled water into a serum bottle filled with a soil sample, adjusting the water content of the soil to 30% of mass water content, sealing the bottle by using an untreated rubber spacer and compacting the bottle by using an aluminum cover, then replacing headspace gas in the bottle by adopting a He flushing mode, filling the serum bottle with high-purity He for 30s after vacuumizing for 300s, filling the bottle with the filling pressure of 1.5MPa for 6 times, finally inserting a 10ml piston-free syringe (containing 2ml distilled water and hermetically isolating atmospheric exchange) into the serum bottle to balance the pressure, and placing the sample in the air to be tested. And measuring by gas chromatography for 15h, 26h, and 39h, wherein the same serum bottle is used at each time point in the measurement, and the bottle N is used 2 Calculating to obtain N according to the linear relation of the concentration changing with time 2 The leak rate of (c). The experiment was performed in 3 replicates, i.e. in a total of 3 serum bottles.
The specific measurement process of the treatment for measurement by using the method provided by the invention is as follows: the method comprises the steps of smearing a small amount of vacuum lubricating silicone grease on a serum bottle opening filled with a soil sample, adding distilled water which is filled with He in advance for 3 hours, adjusting the water content of soil to 30% of the mass water content, washing the serum bottle opening with He in advance, sealing the serum bottle opening with a rubber spacer which is placed in the He environment, compacting the serum bottle opening with an aluminum cover, replacing headspace gas in the bottle in a He washing mode, filling the serum bottle with high-purity He for 30 seconds after vacuumizing for 300 seconds, repeatedly performing the filling process for 6 times at the filling pressure of 1.5MPa, inserting a 10ml piston-free injector (containing 2ml of distilled water and hermetically isolating atmospheric exchange) into the serum bottle to balance the pressure, and placing the serum bottle opening in an anaerobic box which takes the He as a gas source for testing. And measured using gas chromatography at 15h, 26h, 39h (it is specifically stated here that the time point for measurement should be 48h since the serum bottle containing the sample according to the method of the invention requires a further incubation for 48h after He rinse, but for better contrast of the method of the invention with the method of the background art, the time for measurement is advanced in the present experiment, i.e. measured from 15 h) the destructive sampling is used in the measurementSample, i.e. each time point was measured using a different serum bottle, each serum bottle was measured only once, using N in the bottle 2 Calculating to obtain N according to the linear relation of the concentration changing with time 2 The leak rate of (c). The experiment was performed in 9 replicates, i.e. a total of 9 serum vials, and three serum vials were randomly selected for each time point.
Calculated to obtain the N of the treatment measured by the background technique 2 The leakage rate was 2.76. Mu.L/L/h and the leakage rate tended to increase gradually with the passage of time (as shown in FIG. 2), and the N was measured by the method of the present invention 2 The leakage rate was 0.30 μ L/L/h and the leakage rate tended to decrease gradually with time (as shown in FIG. 3), and N in the serum bottle at 39h 2 Concentration and N in serum bottle at 26h 2 The concentration differed by only 0.23. Mu.L/L. The reason for the difference between the two methods is that neither the rubber septum used for the closure nor the distilled water added to the serum bottle of the background art is responsible for the N contained therein 2 The removal is performed while the head gas in the serum bottle is a low N 2 Environment, thus N adsorbed by the rubber spacers 2 With N dissolved in distilled water 2 Will gradually be released towards the headspace. In addition, the background technology adopts non-destructive sampling, and the sealing performance of the rubber spacer used for sealing the serum bottle can be gradually destroyed along with the increase of the sampling times, so that the leakage rate is large and tends to gradually increase along with the time when the background technology is used for measurement. In the method provided by the invention, the sealing performance of the rubber spacer can be effectively protected by adopting destructive sampling, and then N adsorbed to the rubber spacer is utilized 2 Dissolving N in distilled water 2 The removal of the nitrogen-containing gas can enable the headspace gas of the serum bottle and the two media to reach balance more quickly, so the method provided by the invention can obviously reduce the N of the serum bottle culture system 2 The leakage rate. As can be seen from FIG. 3, in the first 39h, a certain amount of N is still present in the serum bottle measured by the method of the present invention 2 Leakage, and because the system has good sealing performance, the main source of leakage of the system is N adsorbed by soil particles and rubber spacers in a serum bottle 2 And N dissolved in distilled water 2 . These N 2 Will gradually release to the head space of the serum bottle with time, and reach equilibrium after a certain time, no longer release, so the leakage rate has the tendency of gradually slowing down with the time. And due to N in the serum bottle at 39h 2 Concentration and N in serum bottle at 26h 2 The concentration differed only by 0.23. Mu.L/L, so N in serum bottles was considered after 39h 2 The leakage rate is not obviously increased any more and can be stabilized at an extremely low level, and will not be applied to N in the experiment 2 The measurement of the discharge rate is influenced, and in order to make the whole serum bottle system reach the stability before the measurement is started as much as possible, the method provided by the invention takes 48 hours after the serum bottle is subjected to He flushing as a starting point for measurement, and ensures that the starting time is longer than the time when the leakage rate of the serum bottle system reaches the equilibrium stability, so the method provided by the invention is used for N 2 The measurement of the discharge rate can lead to more accurate results.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (4)
1. Dry land soil background N 2 A method of determining a discharge rate, comprising the steps of:
step 1, adding 30g of air-dried soil into a 120ml serum bottle;
step 2, treating the rubber spacer by using high-purity He with the purity of 99.999% in a He flushing mode to enable the rubber spacer to be placed in a He environment in advance;
the concrete step of treating the rubber spacer in the He flushing mode in the step 2 comprises the following steps:
(1) Putting 20 rubber spacers into a 120ml cylindrical bottle, sealing, and flushing the cylindrical bottle by using He, specifically, vacuumizing the cylindrical bottle for 300s, then using high-purity He with the purity of 99.999 percent, filling for 30s, and performing filling pressure of 1.5MPa repeatedly for 6 times;
(2) Placing for 24h, performing He flushing treatment again, and continuing to place for 24h for later use;
step 3, regulating the water content of the soil sample in the serum bottle by using distilled water to ensure that the water content of the soil sample is 30 mass percent;
step 4, coating a small amount of vacuum lubricating silicone grease on the opening of the serum bottle, sealing the opening by using a rubber spacer which is placed in the He environment in advance, and compacting by using an aluminum cover;
step 5, replacing headspace gas in the bottle by adopting a He flushing mode;
the method specifically comprises the steps of vacuumizing a serum bottle for 300s, filling high-purity He with the purity of 99.999% for 30s under the filling pressure of 1.5MPa for 6 times;
step 6, using a 10ml piston-free injector to insert into a serum bottle to balance the pressure;
step 7, placing the serum bottle into an anaerobic box to reduce the external N 2 Coating a layer of 704 sealing silica gel outside the rubber spacer to further prevent the leakage of outside air, and continuously culturing for 48h;
in the step 7, the anaerobic tank uses He as a gas source, the purity of the He is 99.999 percent, and low N is built in the tank body 2 An environment;
step 8. The headspace gas of the serum bottle was measured at time intervals using gas chromatography.
2. Dry land soil background N according to claim 1 2 The method for measuring the discharge rate is characterized in that the distilled water used for adjusting the water content of the soil sample in the step 3 needs to be filled with He in advance for 3 hours, and the He purity is 99.999 percent.
3. Dry land soil background N according to claim 1 2 The method for measuring the discharge rate is characterized in that in the step 6, 2ml of distilled water is contained in a 10ml piston-free syringe which is sealed and isolated from airStatus.
4. Dry land soil background N according to claim 1 2 The method for determining the discharge rate is characterized in that the step 8 also comprises sampling in a destructive sampling mode, namely, more parallel processing is set in each group, and all serum bottles are sampled and determined once.
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