CN111562331A - Nitrogen fixation enzyme activity determination device improved by acetylene reduction method and determination method thereof - Google Patents

Nitrogen fixation enzyme activity determination device improved by acetylene reduction method and determination method thereof Download PDF

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Publication number
CN111562331A
CN111562331A CN202010520360.XA CN202010520360A CN111562331A CN 111562331 A CN111562331 A CN 111562331A CN 202010520360 A CN202010520360 A CN 202010520360A CN 111562331 A CN111562331 A CN 111562331A
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valve
gas
rubber plug
nutrient solution
sealed reaction
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李冰
赵俊雯
张瑞平
陶琦
王昌全
张宗锦
李一丁
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Panzhihua Corp Of Sichuan Province Tobacco Monopoly Administration
Sichuan Agricultural University
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Panzhihua Corp Of Sichuan Province Tobacco Monopoly Administration
Sichuan Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection

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Abstract

The invention discloses a device and a method for measuring the activity of nitrogenase improved by using an acetylene reduction method, wherein the device comprises a sealed reaction bottle, an isobutyl rubber plug is arranged at the mouth of the sealed reaction bottle, a gas sampling tube and a nutrient solution sampling tube are fixed on the isobutyl rubber plug, the gas sampling tube is sequentially connected with a first valve and a second valve, and the other end of the gas sampling tube is communicated with an injector through a rubber plug; the nutrient solution sampling pipe is sequentially provided with a third valve and a multi-connection pipe valve; the multi-connection pipe valve is connected with a nutrient solution bottle; the sealed reaction bottle is positioned in the porous heat-preservation type vibration plastic component. The invention solves the problem of air leakage caused by pinholes in the process of measuring the activity of azotase, reduces the system error of each reaction system and improves the reproducibility of experimental results; the full and thorough reaction of the substances is facilitated; meanwhile, due to the design of the multi-joint valve, an operator can finish more sample pretreatment in batches within a certain time, and the experimental efficiency is improved.

Description

Nitrogen fixation enzyme activity determination device improved by acetylene reduction method and determination method thereof
Technical Field
The invention relates to the field of testing method and device improvement, in particular to a nitrogenase activity measuring device improved by an acetylene reduction method and a measuring method thereof, which are used for accurately and efficiently measuring the nitrogenase activity.
Background
The acetylene reduction method is one of the commonly used detection methods, and the method is to indirectly determine whether the azotobacter activity exists and the level of the azotobacter activity by detecting whether acetylene is reduced to form ethylene and the amount of the ethylene through a gas chromatograph according to the principle that the azotobacter has substrate diversity. At present, most of the activities of nitrogen-fixing enzymes of soil or plant nodules are measured by an acetylene reduction method in laboratories.
The general method of the experiment is that a needle tube with a needle head is inserted into a bottle mouth with serum, 1ml of mixed gas is pumped, and a gas chromatograph is adopted to measure ethylene (C)2H4) And calculating the nitrogenase activity according to the formula. The rubber plug of the serum bottle is pricked by the needle tube with the needle for a plurality of times, thereby realizing a plurality of times of measurement. The method can aggravate the damage of the rubber plug and cause the air leakage of the device, thereby causing the problems of inaccurate experimental data, poor reproducibility and the like. Meanwhile, the experiment requires that an experimenter can only finish one experimental sample from sample pretreatment to final analysis and determination of a reactant at one time, and the experiment has the advantages of long time consumption, low experiment efficiency and large system error caused by temperature change and manpower. Aiming at the problems, reducing the air leakage of a reaction device and the entrance of external air in the experimental process and improving the pretreatment efficiency of a sample are important directions for optimizing the determination procedure of the azotase activity, but the prior art is lack of correspondingAn apparatus and method.
Disclosure of Invention
The purpose of the invention is as follows: the optimized system and method for determining the azotase activity by using the acetylene reduction method are provided, and are used for accurately and efficiently measuring the azotase activity of soil and nodules.
The technical scheme of the invention is as follows:
the azotase activity measuring device improved by the acetylene reduction method comprises a sealed reaction bottle, wherein an isobutyl rubber plug is arranged at the mouth of the sealed reaction bottle, a gas sampling tube and a nutrient solution sampling tube are fixed on the isobutyl rubber plug, the gas sampling tube is sequentially connected with a first valve and a second valve, and the other end of the gas sampling tube is communicated with an injector through a rubber plug;
the nutrient solution sampling pipe is sequentially provided with a third valve and a multi-connection pipe valve; the multi-connection pipe valve is connected with a nutrient solution bottle;
the sealed reaction bottle is positioned in the porous heat-preservation type vibration plastic component.
The inside of the sealed reaction bottle is filled with a sample needing to be measured for the activity of the azotobacter; the porous heat-preservation type vibration plastic component is used for placing a sealed reaction bottle in a vibration incubator; the porous heat-preservation type vibration plastic component can be used for placing a plurality of reaction bottles at the same time, and is convenient to take; meanwhile, the temperature can be kept to reduce the temperature difference in each reaction bottle in the experiment; the same-frequency oscillation can promote the reaction to be more sufficient and thorough, and reduce the system error.
The multi-connection pipe valve can connect a plurality of sample nutrient solution sampling pipes together and input nutrient solution simultaneously.
The invention also provides a method for measuring the activity of the nitrogenase improved by using the acetylene reduction method, and the device for measuring the activity of the nitrogenase improved by using the acetylene reduction method comprises the following steps:
A) taking 1g of fresh soil or plant root nodule 10g, and filling into a 100ml sealed reaction bottle;
B) covering an isobutyl rubber plug with a nutrient solution sampling pipe and a gas sampling pipe;
C) 2ml of 0.1mol/l glucose nutrient solution is added into the sealed reaction bottle through a nutrient solution sampling tube, and a third valve is closed;
D) connecting the syringe with a rubber plug, opening the second valve, keeping the first valve closed, and sucking air in the gas sample introduction pipe until the pipe is shriveled;
E) closing the second valve, taking down the syringe to discharge air in the tube, and connecting the rubber plug again;
F) simultaneously opening the first valve and the second valve, sucking 10ml of air, closing the first valve and the second valve, and taking down the syringe to discharge the air in the tube;
G) pumping 10ml of acetylene gas with the purity of 99.9 percent by using an injector, connecting a rubber plug, sequentially opening a second valve and a first valve of a valve, injecting the gas into a sealed reaction bottle, and then rapidly closing the second valve and the first valve of the valve;
H) placing the sealed reaction bottle in a porous heat-preservation type vibration plastic component, and culturing for 24 hours in a constant-temperature vibration incubator at 28 ℃;
I) opening the second valve, using the injector to suck to discharge the air in the gas sampling pipe, connecting the rubber plug again, and opening the first valve to suck 10ml of reaction gas for determination;
J) ethylene (C) was determined by gas chromatography (GC, Agilent7890B, USA)2H4) The gas chromatograph adopts a capillary column (DB-WAX, 122-; and (4) calculating the nitrogen fixation activity of the soil by using the equation (1) according to the ethylene content measured in the result.
Figure BDA0002531848610000021
Wherein: ARA: nitrogenase activity, expressed in moles of ethylene produced per unit of soil per unit of time; k is the ethylene molar quantity, mu mol; m issoilVolume of moist soil, g; h isreactionFor incubation time, d;
Figure BDA0002531848610000022
volume of ethylene sample, ml; atoms represent the pressure generated by the mercury column with the corresponding height of the experimental environment air pressure, and mm and Hg are common pressure units; 760mm Hg isA common pressure value, i.e. a standard atmospheric pressure; t: temperature of experimental environment, DEG C.
Before injecting acetylene gas into reaction system, connect the rubber buffer with the syringe, open the second valve and close first valve, the intraductal air of suction gas sampling becomes shrivelled in order to reduce reaction system foreign gas to the pipe, improves experimental result accuracy and degree of accuracy.
Can combine the timing function of the incubator to obtain the measured ethylene (C)2H4) Yield to obtain nitrogenase activity (rate of ethylene reduction per unit time of soil/nodule) data.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention can simultaneously carry out the pretreatment of a plurality of samples, and avoids the complicated operation of pricking holes and adding nutrient solution by adopting the traditional injector.
(2) The invention can directly realize the closing of the pipeline through the valve of the PVC hose, realize the communication between the serum bottle and the injector, reduce the influence of the air leakage of the device on a reaction system caused by the damage of the rubber plug due to the pricking of the needle tube with the needle head, and improve the accuracy and the reproducibility of experimental data.
(3) The system has simple structure, accurate measurement result, high test speed and low cost, and provides favorable help for analyzing and researching azotase, microbial gas production degree, azotobacter efficiency and the like.
Drawings
FIG. 1 is a schematic perspective view of the present invention;
FIG. 2 is a schematic structural view of the present invention;
FIG. 3 is a schematic view of a porous insulating oscillating plastic component according to the present invention.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
The first embodiment is as follows:
as shown in fig. 1 and 2, the device for measuring the activity of a nitrogenase improved by using an acetylene reduction method comprises a sealed reaction bottle 7, wherein an isobutyl rubber plug 6 is arranged at the mouth of the sealed reaction bottle 7, a gas sampling tube 4 and a nutrient solution sampling tube 9 are fixed on the isobutyl rubber plug 6, the gas sampling tube 4 is sequentially connected with a first valve 5 and a second valve 3, and the other end of the gas sampling tube 4 is communicated with an injector 1 through a rubber plug 2;
the nutrient solution sampling pipe 9 is sequentially provided with a third valve 10 and a multi-connection pipe valve 11; the multi-joint valve 11 is connected with a nutrient solution bottle 12;
the sealed reaction bottle 7 is positioned in the porous heat-preservation type vibration plastic component 8.
A sample needing to be measured for the activity of the azotobacter is contained in the sealed reaction bottle 7; the porous heat preservation type oscillation plastic component 8 is used for placing a sealed reaction bottle in an oscillation incubator, as shown in figure 3; the multi-joint valve 11 can connect a plurality of sample nutrient solution feeding pipes together and simultaneously feed nutrient solution. The nutrient solution sampling pipe 9 is a PVC hose with the diameter of only 1mm, so that the residue of gas or liquid on the pipe wall can be effectively reduced, and the experimental accuracy is improved.
The gas sample inlet pipe 4 of the injector 1 connected with the sealed reaction bottle 7 is a PVC hose with the diameter of 1 mm; the device comprises two gas stop valves, wherein the second valve 3 close to the injector 1 is opened, and the first valve 5 close to the sealed reaction bottle 7 is closed, so that impurity gas in the device system can be effectively discharged; the gas replacement can be accurately carried out by opening the first valve 5 and the second valve 3 simultaneously; the first valve 5 and the second valve 3 are closed to realize the systematic sealing of the azotase activity measuring device, so that the influence of the air leakage of the device caused by the damage of the rubber plug 2 due to the fact that the needle tube with the needle head is used for pricking the rubber plug is reduced, and the experimental accuracy is further improved.
The syringe 1 for sucking gas should be exclusively used for gas.
Example two:
as shown in fig. 1, a method for measuring the activity of nitrogenase improved by the acetylene reduction method according to the present invention is as follows: the measuring system is used for a azotase activity determination experiment in a common laboratory, a serum bottle with the volume of 100ml is selected as a sealed reaction bottle 7, a PVC (polyvinyl chloride) catheter with the diameter of 1mm is selected, a nutrient solution bottle 12 is selected as a washing bottle with scales, selected air stop valves, namely a first valve 5, a second valve 3 and a third valve 10 are spiral air stop valves, a capillary column is selected as DB-WAX, 122-7032, Agilent, USA, and the range of a selected injector 1 is 15 ml.
The specific operation steps are as follows:
soil or nodule sample preparation:
soil sample: and selecting collected fresh soil, and removing impurities such as residual gravels, animal residues, plant roots and the like in the soil. 10.00g of soil was carefully weighed into a 100ml sealed reaction flask 7.
Root nodule sample: the leguminous plant is dug out from the field, the overground part is cut off, the root is cleaned and dried slightly, and the root part with the nodulation is cut off respectively. The nodules on the main roots can be directly peeled off, a small number of roots on the nodules on the lateral roots are cut off, fresh nodules are weighed, and 1g of fresh nodules are taken and sent into a 100ml sealed reaction flask 7.
(1) Preparing nutrient solution and experimental gas:
preparing 0.1mol/l glucose nutrient solution: weighing 18.01g of glucose, placing into a beaker, adding a little distilled water, stirring by a glass rod until the glucose is dissolved, and introducing a 1000ml nutrient solution bottle 12 to a constant volume for standby.
Acetylene gas: purity 99.99%, purchased from Chengdu Ruitai gas Co.
Standard ethylene gas: purity 99.99%, purchased from Chengdu Ruitai gas Co.
Sample pretreatment:
two groups of soil azotase activity determination samples are set: [ IMPROVED GROUP ] is to use the improved system to carry on the azotase determination, and [ CONTROLL GROUP ] is to use the traditional method to carry on the azotase determination.
Note: the method takes a soil sample as an example for operation demonstration, and the method is consistent when the nodule sample is measured.
[ improvement group ]: a100 ml sealed reaction bottle 7 with a 1g soil sample is covered by an isobutyl rubber plug 6 with a nutrient solution sampling tube 9 and a gas sampling tube 4 for sealing, and all valves are closed at the moment to form the sealed reaction bottle 7. The third valve 10 is opened, 2ml of 0.1mol/l glucose nutrient solution is added into the sealed reaction flask 7 through the nutrient solution sampling tube 9, and the third valve 10 is closed. Then, the syringe 1 is connected with the rubber plug 2, the second valve 3 is opened, the first valve 5 is closed, and air in the gas sampling tube 4 is sucked until the tube is deflated; closing the second valve 3, taking down the syringe 1 to discharge air in the tube, and connecting the rubber plug 2 again; simultaneously opening the second valve 3 and the first valve 5, sucking 10ml of air, closing the second valve 3 and the first valve 5, and taking down the syringe 1 to discharge air in the tube; pumping 10ml of acetylene gas with the purity of 99.9 percent by using a syringe 1, connecting a rubber plug 2, sequentially opening a second valve 3 and a first valve 5, injecting the acetylene gas into a sealed reaction bottle 7, then rapidly closing the first valve 5 and the second valve 3 of the valves, and sealing the bottle mouth by using a Parafilm sealing film;
[ control group ]: a100 ml serum bottle with 1g soil sample is taken, 2ml of 0.1mol/l glucose nutrient solution is added, and an isobutyl rubber plug is covered. The rubber stopper was pricked with a 20ml syringe, and after taking the air in the 10ml bottle, another syringe was used to inject 10ml of acetylene gas into the serum bottle. The Parafilm sealing film seals the bottle mouth.
(2) Culture experiment:
[ improvement group ]: the sealed reaction bottle 7 is placed in a porous heat-preservation type vibration plastic component 8, and is placed in a constant-temperature vibration incubator together, the temperature is set to be 28 ℃, the time is 24 hours, and the rotating speed is 150 rpm. And taking out the sealed reaction bottle 7 after the culture time is over.
[ control group ]: the serum bottle was placed in a common constant temperature incubator and incubated at 28 ℃ for 24 h. And taking out the serum bottle after the culture time is over.
(3) Analysis and test:
[ improvement group ]: connect syringe 1 and rubber buffer 2, open second valve 3, use syringe 1 to aspirate and advance the interior air of pipe 4 with exhaust gas, connect rubber buffer 2 again, open first valve 5 and aspirate 1ml reaction gas respectively and insert the gas chromatograph inlet. Two-group system ethylene (C) was determined by gas chromatography (GC, Agilent7890B, USA)2H4) The gas chromatograph adopts a capillary column (DB-WAX, 122-. From the measured ethylene content in the results, the soil nitrogen fixation activity (ARA) was calculated using equation (1).
Figure BDA0002531848610000051
Wherein: ARA: nitrogenase activity, expressed in moles of ethylene produced per unit of soil per unit of time; k is the ethylene molar quantity, mu mol; m issoilVolume of moist soil, g; h isreactionFor incubation time, d;
Figure BDA0002531848610000052
volume of ethylene sample, ml; atoms represent the pressure generated by the mercury column with the corresponding height of the experimental environment air pressure, and mm and Hg are common pressure units; 760mm Hg is a common pressure value, i.e., one standard atmosphere; t: temperature of experimental environment, DEG C.
[ control group ]: the rubber stopper of the serum bottle is pricked by using a syringe, and gas in the 1ml bottle is pumped into a sample inlet of the gas chromatograph. Two-group system ethylene (C) was determined by gas chromatography (GC, Agilent7890B, USA)2H4) The gas chromatograph adopts a capillary column (DB-WAX, 122-. From the measured ethylene content in the results, the soil nitrogen fixation activity (ARA) was calculated using equation (1).
(4) And (3) test results:
under the same conditions of culture temperature and culture time, the nitrogen-fixing enzyme activity of the soil sample of the improved group in the experiment is slightly higher than that of the control group. Compared with the control group, the nitrogen-fixing enzyme activity of the soil sample in the improved group is averagely higher by 7.00 percent. Three aspirations were performed at intervals of 10 minutes on the same reaction flask, and the results showed that the deviation of the results of the control group was 36.76%, 66.74% and 81.68%; the deviation of the results of the three times of the improved group is 4.64 percent, 8.01 percent and 15.08 percent, and the results of the three times of the repeated measurement are not significantly different. The results of this study show that the improved method described herein allows for more accurate determination of soil nitrogenase activity with reduced deviation in results.
TABLE 1 Activity and reproducibility of soil azotobacter after cultivation
Figure BDA0002531848610000061
The above embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and various modifications and changes may be made by those skilled in the art. All equivalent substitutions or modifications made according to the technical scheme and the inventive concept of the present invention within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. The azotase activity measuring device improved by using an acetylene reduction method is characterized by comprising a sealed reaction bottle (7), wherein an isobutyl rubber plug (6) is arranged at the mouth of the sealed reaction bottle (7), a gas sample inlet pipe (4) and a nutrient solution sample inlet pipe (9) are fixed on the isobutyl rubber plug (6), the gas sample inlet pipe (4) is sequentially connected with a first valve (5) and a second valve (3), and the other end of the gas sample inlet pipe (4) is communicated with an injector (1) through a rubber plug (2);
the nutrient solution sampling pipe (9) is sequentially provided with a third valve (10) and a multi-joint pipe valve (11); the multi-joint pipe valve (11) is connected with a nutrient solution bottle (12);
the sealed reaction bottle (7) is positioned in the porous heat-preservation type vibration plastic component (8).
2. A method for measuring an activity of a nitrogenase improved by an acetylene reduction method, comprising the steps of:
A) fresh soil or plant root nodules are taken and put into a sealed reaction bottle (7);
B) an isobutyl rubber plug (6) with a nutrient solution sampling pipe (9) and a gas sampling pipe (4) is covered;
C) adding glucose nutrient solution into the sealed reaction bottle (7) through the nutrient solution sampling tube (9), and closing the third valve (10);
D) connecting the syringe (1) with the rubber plug (2), opening the second valve (3), keeping the first valve (5) closed, and pumping air in the gas sampling tube (4) until the tube is shriveled;
E) closing the second valve (3), taking down the syringe (1) to discharge air in the tube, and connecting the rubber plug (2) again;
F) simultaneously opening the first valve (5) and the second valve (3), closing the first valve (5) and the second valve (3) after sucking a certain volume of air, and taking down the injector (1) to discharge air in the pipe;
G) using an injector (1) to suck acetylene gas with a certain volume purity of 99.9%, connecting a rubber plug (2), sequentially opening a second valve (3) and a first valve (5), injecting the gas into a sealed reaction bottle (7), and then rapidly closing the second valve (3) and the first valve (5);
H) placing the sealed reaction bottle (7) in a porous heat-preservation type vibration plastic component (8), and culturing for 24 hours in a constant-temperature vibration incubator at 28 ℃;
I) opening the second valve (3), using the injector (1) to suck to exhaust air in the gas sampling pipe (4), connecting the rubber plug (2) again, and opening the first valve (5) to suck reaction gas for determination;
J) measuring the ethylene production by using a gas chromatograph; and (3) calculating the nitrogen fixation activity of the soil by using an equation (1) according to the ethylene content measured in the result:
Figure FDA0002531848600000011
wherein: ARA: nitrogenase activity, expressed in moles of ethylene produced per unit of soil per unit of time; k is the ethylene molar quantity, mu mol; m issoilVolume of moist soil, g; h isreactionFor incubation time, d;
Figure FDA0002531848600000012
volume of ethylene sample, ml; atoms represent the pressure generated by the mercury column with the corresponding height of the experimental environment air pressure, and mm and Hg are common pressure units; 760mmHg is a common pressure value, i.e., one standard atmosphere; t: temperature of experimental environment, DEG C.
CN202010520360.XA 2020-06-10 2020-06-10 Nitrogen fixation enzyme activity determination device improved by acetylene reduction method and determination method thereof Pending CN111562331A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112305101A (en) * 2020-10-19 2021-02-02 江西省林业科学院 Method for determining nitrogen fixation activity of monochamus alternatus living body

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112305101A (en) * 2020-10-19 2021-02-02 江西省林业科学院 Method for determining nitrogen fixation activity of monochamus alternatus living body

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