MICROCOSMIC CULTURE DEVICE AND ITS APPLICATION IN QUANTITATIVE ANALYSIS OF SOIL CARBON DIFFUSION AND MICROBIAL UTILIZATION PROCESSES

Abstract

The invention relates to the field of soil process analysis, in particular to a microcosmic culture device and its application in quantitative analysis of soil carbon diffusion and microbial utilization process. The microcosmic culture device comprises a closed container, an incubator and a dialysis tube in the closed container; The incubator comprises a soil layer; The dialysis tube is connected with the incubator, and part of the tube extends through the side wall of the incubator into the soil layer along the length direction. The dialysis tube is equipped with a carbon source, and the dialysis tube can make the carbon source spread to the soil layer, and always maintain the same water potential inside and outside the dialysis tube. The invention provides a quantitative analysis method for soil carbon diffusion and microbial utilization process based on the microcosmic culture device, through which the relationship between the efficiency of microbial utilization of exogenous carbon source and space can be explored, and the influence of the distance of carbon diffusion on the efficiency of microbial utilization of exogenous carbon can be further quantitatively analyzed.

Claims

1. A microcosmic culture device for quantitative analysis of soil carbon diffusion or microbial utilization processes, comprising: a closed container, an incubator and a dialysis tube in the closed container; wherein the incubator comprises a soil layer in which soil is evenly laid; the dialysis tube is connected with the incubator, and part of a tube body of the dialysis tube extends into the soil layer along a length direction through side walls of two opposite sides of the incubator, and the dialysis tube is parallel with two unpenetrated side walls of the incubator and has the same distance with the two unpenetrated side walls; the dialysis tube is made of a selective dialysis membrane with a threshold size of 12-14kD, the dialysis tube is equipped with a carbon source which can be diffused to the soil layer, dextran is added to the dialysis tube as a microcirculation dredge agent to ensure that water potential in the dialysis tube is consistent with that in soil solution.

2. The microcosmic culture device according to claim 1, wherein the side walls of the incubator is a sterile plate.

3. Use of the microcosmic culture device of claim 1 in quantitative analysis of soil carbon diffusion or microbial utilization processes.

4. Use of the microcosmic culture device according to claim 3, comprising cultivating microorganisms using the microcosmic culture device and quantitatively analyzing soil carbon diffusion or microbial utilization processes through changes in CO.sub.2 concentrations in air and soil in the closed container.

5. Use of the microcosmic culture device according to claim 4, comprising setting up a treatment group and a control group, the dialysis tube in the treatment group is loaded with glucose or .sup.14C-glucose, and the dialysis tube in the control group is not loaded with carbon source; soil samples with different distances from dialysis tubes are obtained and microbial biomass carbon content is detected; the process of soil carbon diffusion or microbial utilization is quantitatively analyzed by determination of microbial biomass carbon from multiple soil samples in the treatment group and the control group.

6. Use of the microcosmic culture device according to claim 5, wherein an amount of microbial biomass carbon in the test is: the amount of microbial biomass carbon in the test is detected by substrate induced respiration or chloroform extraction.

7. Use of the microcosmic culture device according to claim 5, the soil samples obtained at different distances from the dialysis tubes are: the soil samples obtained at different distances from the dialysis tube are obtained at fixed distances of 0.25 to 1 cm.

8. Use of the microcosmic culture device of claim 2 in quantitative analysis of soil carbon diffusion or microbial utilization processes.

9. Use of the microcosmic culture device according to claim 8, comprising cultivating microorganisms using the microcosmic culture device and quantitatively analyzing soil carbon diffusion or microbial utilization processes through changes in CO.sub.2 concentrations in air and soil in the closed container.

10. Use of the microcosmic culture device according to claim 6, the soil samples obtained at different distances from the dialysis tubes are: the soil samples obtained at different distances from the dialysis tube are obtained at fixed distances of 0.25 to 1 cm.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1 provides a microcosmic culture device for embodiment 1 of the invention;

[0036] In FIG. 1: 1. Closed container; 2. Incubator; 3. Dialysis tube; 4. Soil layer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure will be further described below with the preferred embodiment, but the present invention is not limited to the following examples.

Embodiment 1

[0038] The invention provides a microcosmic culture device, as shown in FIG. 1, which comprises a closed container 1, an incubator 2 and a dialysis tube 3 in the closed container 1;

[0039] The incubator 2 comprises a soil layer 4;

[0040] The dialysis tube 3 is connected with the incubator 2, and part of the tube body extends along the length through the side wall of the incubator 2 into the soil layer 4.

[0041] Airtight container 1 can be selected from a variety of airtight containers commonly used in this field, as long as the air tightness is maintained, such as a capped wide-mouth bottle.

[0042] Further, the dialysis tube 3 passes through two side walls of the incubator 2;

[0043] Preferably, the dialysis tube 3 passes through the lateral walls of two opposite sides of the incubator 2;

[0044] Further preferably, the dialysis tube 3 is parallel to the untraversed side wall and is the same distance from both untraversed side walls. In the case of the same distance, other factors except spatial distance are ensured to be relatively unchanged, making it easier to control other variables to ensure the accuracy of exploring the efficiency of microorganisms' utilization of exogenous carbon sources and the spatial relationship.

[0045] Further, the side wall of the incubator 2 is a sterile plate to prevent the influence of miscellaneous bacteria on the experimental results.

[0046] Further, the soil in the soil layer 3 is evenly laid.

[0047] In practical application, the microcosmic culture device can be used for quantitative analysis of soil carbon diffusion or microbial utilization process, specifically including:

[0048] The microcosmic culture device was used for microbial culture, and the treatment group and the control group were set up. Glucose or .sup.14C-glucose was loaded into the dialysis tube in the treatment group, and no carbon source was added into the dialysis tube in the control group.

[0049] Soil samples with different distances from dialysis tubes were obtained and microbial biomass carbon content was detected.

[0050] The process of soil carbon diffusion or microbial utilization was quantitatively analyzed by the determination of microbial biomass carbon from multiple soil samples in the treatment group and the control group.

[0051] Among them, the soil samples with different distances from the dialysis tube can be obtained in various ways, such as 0.5 cm or 1.0 cm as spacing, 0-0.5 cm, 0.5-1 cm, 1.0-2.0 cm, random sampling at each spatial location at least 5 times, evenly mixed samples as representative of the spatial location of the sample.

Embodiment 2

[0052] Based on the microcosmic culture device provided in Embodiment 1, this embodiment provides a quantitative analysis method for soil carbon diffusion and microbial utilization processes, specifically including the following flow:

[0053] The bulk density and field water capacity of some dryland soil samples were measured. Plant residues and small stones were removed from the remaining soil samples and ground to a 2 mm screen to obtain the soil for test. Soil pH, carbon and nitrogen, water content and the bulk density of the original soil were measured. Deionized water was added to make the soil moisture content 65% of the field water capacity. The water potential measurement system was used to measure the soil water potential. According to the soil water potential, the increment of Dextran in the dialysis tube (made of a selective dialysis membrane with a threshold size of 12-14kD) was calculated, and the increment of dextran was added, specifically referring to the water potential of the solution of ψ.sub.DEX=−22.5[DEX].sup.2−1.4[DEX] (ψ.sub.DEX, Dextran 40; [DEX], Dextran 40 solution concentration). Three treatments were set up. The first was loaded into the dialysis tube with ordinary glucose, the second was loaded into the dialysis tube with .sup.14C-glucose, and the third was loaded into the dialysis tube without carbon source (control group). The experiment was carried out according to the following steps:

[0054] 1. CO.sub.2 test

[0055] The dialysis tube was placed in the center of the incubator, soil was evenly laid on both sides, and the incubator was surrounded by sterile boards. The completed incubator was placed in a sterile wide-mouth bottle and sealed for culture. Culture for 8 days, during which the concentration of CO.sub.2 or .sup.14C-CO.sub.2 in the bottle was monitored in real time.

[0056] 2. Substrate induced respiration method to detect the amount of microbial biomass activated carbon

[0057] The dialysis tube was placed in the center of the incubator, soil was evenly laid on both sides, and the incubator was surrounded by sterile boards. The completed incubator was placed in a sterile wide-mouth bottle and sealed for culture.

[0058] The above culture devices were equipped with multiple devices, and the open-cover sampling of any device was randomly selected regularly. The sampling standard was divided into three sub-samples according to the distance from the dialysis tube: 0-0.5 cm soil sample, 0.5-1.0 cm soil sample and 1.0-2.0cm soil sample. The substrate-induced respiration method was used to determine the microbial biomass activated carbon of each soil sample, specifically as follows: The mixture was thoroughly mixed with 8 g fresh soil/20 ml yeast solution and cultured in a closed sterilized bottle, during which the oscillations were reciprocated at 180 rpm. At 0, 30, 60, 120 and 180 minutes, the gas in the bottle was collected by syringe and CO.sub.2 concentration was immediately determined by infrared gas analyzer (Li820, Licor Biosciences), and then converted into microbial biomass activated carbon by linear regression analysis.

[0059] 3. Chloroform extraction method to detect the amount of microbial biomass activated carbon

[0060] The dialysis tube was placed in the center of the incubator, soil was evenly laid on both sides, and the incubator was surrounded by sterile boards. The completed incubator was placed in a sterile wide-mouth bottle and sealed for culture.

[0061] The above culture devices were equipped with multiple devices, and the open-cover sampling of any device was randomly selected regularly. The sampling standard was divided into three sub-samples according to the distance from the dialysis tube: 0-0.5 cm soil sample, 0.5-1.0 cm soil sample and 1.0-2.0 cm soil sample. Chloroform extraction method was used to determine the microbial biomass carbon of each soil sample, specifically as follows: The comparative treatment with chloroform and without chloroform was set. The liquid to be tested was obtained through 30 minutes of chloroform extraction, glass fiber filtration, compressed air bubble removal and other steps. After freezing, the total organic carbon was determined by Shimadzu TOC-V. The group treated with chloroform minus the group treated without chloroform was then converted into microbial biomass carbon by relevant parameters.

[0062] 4. Explanation of experimental results:

[0063] (1) CO.sub.2 test

[0064] In the 8-day culture experiment, the CO.sub.2 emission curve of the control group was y=0.1865×−0.0452 (R.sup.2=0.9816), and that of the carbon source group was y=0.2219×−0.0719 (R.sup.2=0.9811). The CO.sub.2 emission rate of the carbon source group was significantly higher than that of the control group. From the second day, CO.sub.2 emissions in the carbon 10 source group were significantly higher than those in the control group, exceeding 13.5%. On the 8th day, the amount of CO.sub.2 in the carbon source group and the control group still did not reach the peak, indicating that there was enough carbon source for microbial utilization, and that the top space of the culture facility was sufficient for accurate measurement of CO.sub.2 value.

[0065] (2) microbial biomass carbon

[0066] The separation and placement of carbon sources from soil did not affect the utilization of foreign carbon sources by soil microorganisms, which was shown in that the microbial biomass carbon in the carbon source group was significantly higher than that in the control group. In addition, soil microorganisms' use of carbon sources presents a distance gradient rule, which can be shown as follows: The increment of 0-0.5 cm soil microbial biomass carbon was 70-106 mg kg.sup.−1, 0.5-1.0 cm soil microbial biomass carbon was 24-38 mg kg.sup.−1, and 1.0-2.0 cm soil microbial biomass carbon was 1.0-4.0 mg kg.sup.−1. It also indicates that the method and culture device of the invention are suitable for the study of carbon diffusion and utilization of microorganisms.

[0067] (3) .sup.14C-Microbial biomass carbon

[0068] The .sup.14C-microbial biomass carbon showed a gradient pattern, and the .sup.14C-microbial biomass carbon closer to the carbon source (0-0.5 cm) was significantly higher than the .sup.14C-microbial biomass carbon farther away (0.5-1.0 cm and 1.0-2.0 cm). Compared with the control group, the microbial biomass carbon was: The increment of .sup.14C-microbial biomass carbon in 0-0.5 cm soil was 0.0110-0.0160nmol, and that in 0.5-1.0 cm soil was 0.0010-0.0021nmol. The increment of .sup.14C-microbial biomass carbon in 1.0-2.0 cm soil was 0.0005-0.0010nmol. This confirms that microorganisms can utilize exogenous carbon sources as described in Result 2, and utilization efficiency is correlated with spatial location. Carbon diffusion distance affects microbial utilization efficiency of carbon.

[0069] Although the invention has been described in detail by the general description and the specific implementation scheme above, it is obvious to the technical personnel in the field that some modifications or improvements can be made on the basis of the invention. Therefore, the modifications or improvements made on the basis of not deviating from the spirit of the invention are within the scope of protection required by the invention.