Method for detecting discharge of submerged plant and / or non-submerged plant attached biological membrane N2O in freshwater lake

Abstract

The invention discloses a method for detecting discharge of submerged plant and / or non-submerged plant carrier attached biofilm N2O in a freshwater lake. The method comprises the following steps: (1) placing a submerged plant and / or non-submerged plant carrier which is collected from a freshwater lake and is attached with a biological membrane in ultrapure water for dark treatment; (2) putting the submerged plants and / or non-submerged plants which are subjected to dark treatment and are attached with the biological membranes into a serum bottle containing a denitrification culture solution, and sealing; (3) adding acetylene into the acetylene inhibitor group to enable the final volume fraction of the acetylene inhibitor group in the serum bottle to reach 10%, and not adding acetylene into the blank group; and (4) exploring the potential N2O net discharge rate, potential N2O reduction rate and potential denitrification rate of the biological membrane through the inhibitor group and the blank group. The invention provides a new thought for researching submerged plant and non-submerged plant carrier biological membranes, and has important significance for researching biological membrane N2O emission in lakes.

Description

Technical field:

[0001] The present invention relates to the field of environmental protection technology, and in particular to a method for detecting N2O emissions from biofilms attached to submerged plants and / or non-submerged plants in freshwater lakes. Background technology:

[0002] There are two typical types of biofilms in freshwater lakes: submerged plant-supported biofilms and non-submerged plant-supported biofilms. Submerged plant biofilms attach to the stems and leaves of submerged plants, while non-submerged plant biofilms attach to non-biological supports such as rocks, sand, dead branches, and plastic. These two types of biofilms have different oxygen transport pathways. Submerged plants serve as the natural substrate for biofilm growth and provide oxygen to the biofilm through photosynthesis. Nutrients and oxygen in the water exhibit a counter-diffusion pattern within the biofilm. Non-biological supports, on the other hand, cannot provide oxygen to the biofilm. Dissolved oxygen and nutrients in the water exhibit a co-diffusion pattern within the biofilm. Therefore, submerged plant-supported and non-submerged plant-supported biofilms have different oxygen transport pathways, which in turn contributes to differences in the microbial community structure of the biofilms. Biofilms attached to submerged plants play a crucial role in the structure and function of freshwater ecosystems. Biofilms regulate nutrient cycling and energy flow within water bodies, and research on using biofilms for wastewater treatment and remediation of polluted water bodies is gaining increasing attention. Biofilms transform nutrients in the aquatic environment, such as ammonia and nitrates. Therefore, biofilm is an important component of lakes.

[0003] N2O is a greenhouse gas with a global warming potential 298 times that of carbon dioxide (CO2). It is also a contributing factor to the depletion of the stratospheric ozone layer. N2O is primarily a byproduct of nitrification and an intermediate in heterotrophic denitrification. Traditional biofilm research primarily involves washing attached biofilms with phosphate buffered saline (PBS, pH 7.4), without considering the preservation of the biofilm structure. This method fails to accurately reflect the biofilm's N2O production. Summary of the invention:

[0004] The present invention solves the problems existing in the prior art and provides a method for detecting N2O emissions from biofilms attached to submerged plants and / or non-submerged plants in freshwater lakes. The method can efficiently and accurately study N2O emissions while protecting the biofilm microbial community.

[0005] The present invention aims to provide a method for detecting N2O emissions from biofilms attached to submerged plants and / or non-submerged plants in freshwater lakes, comprising the following steps:

[0006] (1) Submerged plants and / or non-submerged plant carriers with biofilms collected from freshwater lakes were placed in ultrapure water for dark treatment;

[0007] (2) Place the submerged plants and / or non-submerged plant carriers with biofilm attached after dark treatment directly into a serum bottle containing denitrification culture medium, seal it, and then purge the gas in the serum bottle with N2 (99.99%) to remove N2O and O2 in the headspace;

[0008] (3) Acetylene was added to the serum bottle to a final volume fraction of 10% (v / v) in the serum bottle, which served as the inhibitor group; the group without acetylene was used as the blank group.

[0009] (4) The potential net N2O emission rate, potential N2O reduction rate, and potential denitrification rate of the biofilm were investigated using the above inhibitor group and blank group. The calculation formula is as follows:

[0010]

[0011] The N2O cumulative concentration is obtained by the following steps: taking out the gas in the experimental group and the blank group at 0-4 hours and detecting the N2O concentration. The present invention uses a gas chromatograph to detect the N2O concentration.

[0012] The method proposed in this paper studies N2O production in biofilms directly on submerged and / or non-submerged plant carriers without disrupting the biofilm microbial community. This method can reveal the status of N2O emissions from submerged and / or non-submerged plant biofilms in lakes. Non-submerged plants include plastic grass, stones, sand, and dead branches.

[0013] The purpose of nitrogen treatment in step (2) is to eliminate O2 and N2O in the serum bottle. The above method is carried out at a temperature of 25±1°C.

[0014] Preferably, the dark treatment in step (1) is carried out for 24-60 hours. More preferably, the dark treatment in step (1) is carried out for 48 hours.

[0015] The purpose of the dark treatment is to eliminate background oxygen, nitrite and nitrate.

[0016] Preferably, the mass volume ratio of the submerged plant and / or non-submerged plant carrier to the denitrification culture solution in step (2) is 1:1.5-2.5 g / mL.

[0017] Further preferably, the mass volume ratio of the submerged plant and / or non-submerged plant carrier to the denitrification culture solution in step (2) is 1:2 g / mL.

[0018] Preferably, the composition ratio of the denitrification culture medium in step (2) is: potassium nitrate 8mg-N / L, glucose 40mg-C / L, potassium dihydrogen phosphate 0.4mg-P / L and trace elements 1ml / L, the pH is adjusted to 7.5 with saturated sodium bicarbonate solution, and the balance is water; the composition ratio of the trace elements is: manganese chloride 1.86g / L, zinc sulfate 0.22g / L, sodium molybdate 0.39g / L, boric acid 2.86g / L, and copper sulfate 0.08g / L. After the denitrification culture medium is prepared, it is deoxygenated by purging with N2 nitrogen for 10 minutes.

[0019] Preferably, the N2O cumulative concentration in step (4) is obtained by taking out 10 mL of gas from the experimental bottle at 0, 0.5 h, 1 h, 2 h, and 4 h, respectively, and detecting the N2O concentration.

[0020] It is further preferred that after taking out 10 mL of gas from the experimental bottle in step (4), 10 mL of nitrogen should be injected to ensure the gas pressure balance in the experimental bottle.

[0021] The present invention also claims the application of the method described herein to studying N2O emissions from biofilms attached to plants and / or non-submerged plant carriers in freshwater lakes. The method proposed herein allows for direct study of biofilm microorganisms attached to the carriers without disrupting the biofilm communities, enabling a more realistic picture of N2O emissions from plant and non-plant biofilms in lakes.

[0022] Compared with the prior art, the present invention has the following advantages: the present invention provides a new idea for studying plant and non-plant carrier biofilms, and is of great significance to the study of N2O emissions from biofilms in lakes. Description of the drawings:

[0023] Figure 1 is the potential net N2O emission rate of the biofilm of Example 1 (attached) and Comparative Example 1 (washed off);

[0024] Figure 2 is the potential N2O reduction rate of the biofilm of Example 1 (attached) and Comparative Example 1 (washed off);

[0025] Figure 3 The potential denitrification rates of the biofilms of Example 1 (attached) and Comparative Example 1 (washed off) are shown. Specific implementation method:

[0026] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0027] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0028] In the following examples, the biofilms used were collected from Vallisneria ovata and small stones attached with biofilms in the plant area and non-plant area of East Lake, Wuhan.

[0029] The denitrification culture medium is composed of 8 mg / L potassium nitrate, 40 mg / L glucose, 0.4 mg / L potassium dihydrogen phosphate, and 1 ml / L trace elements. The pH is adjusted to 7.5 with saturated sodium bicarbonate solution, with the balance being water. The trace element composition is 1.86 g / L manganese chloride, 0.22 g / L zinc sulfate, 0.39 g / L sodium molybdate, 2.86 g / L boric acid, and 0.08 g / L copper sulfate. The culture medium is aerated with 99.99% nitrogen for five minutes to ensure an anaerobic environment.

[0030] The calculation formulas for potential net N2O emission rate, potential N2O reduction rate and potential denitrification rate are as follows:

[0031]

[0032] Example 1

[0033] A method for detecting N2O emissions from plant and / or non-plant carrier-attached biofilms in freshwater lakes comprises the following steps:

[0034] (1) The collected Vallisneria and / or small stones with biofilm attached were placed in darkness for 48 hours.

[0035] (2) Approximately 5 g of Vallisneria ovata and / or small stones with attached biofilm after dark treatment were directly placed in a 120 mL serum bottle containing 10 mL of denitrification culture medium and sealed with a rubber stopper and an aluminum cap.

[0036] (3) Purge the 120 mL serum bottle with nitrogen for 10 minutes to eliminate O2 and N2O in the bottle.

[0037] (4) 12 mL of acetylene was added to the acetylene inhibitor group to a final concentration of 10% (v / v), while no acetylene was added to the blank group.

[0038] (5) For the serum bottles of the blank group and the experimental group, 10 mL of gas was taken out using a syringe with a three-way valve at 0, 0.5 h, 1 h, 2 h, and 4 h, and the concentration of N2O was measured. After taking out 10 mL of gas from the serum bottle, 10 mL of nitrogen was injected to ensure the gas pressure balance in the serum bottle.

[0039] Comparative Example 1

[0040] The same method as in Example 1, except that in step (2), the Vallisneria and / or small stones with biofilm attached were placed in 300 mL of phosphate buffered saline (PBS, pH = 7.4), and then subjected to ultrasound (10 minutes), shaking (225 rpm), and ultrasound (10 minutes), followed by gently scraping off the firmly attached biofilm with a sterile surgical blade. The biofilm suspension was centrifuged and the supernatant was discarded to obtain the biofilm. The biofilm was added to a 120 mL serum bottle. The remaining operations were the same as in Example 1.

[0041] After the above treatment, the potential net N2O emission rate, potential N2O reduction rate and denitrification rate of the submerged plant and non-submerged plant carrier biofilms in the attached state were higher than those of the washed biofilms (such as Figure 1-3 This indicates that the attached biofilm effectively protects the structure of the microbial community, while the washed-off biofilm underestimates N2O emissions.

[0042] Example 2

[0043] The method is the same as Example 1, except that: in step (1), the dark treatment is carried out for 24 hours; in step (2), the mass volume ratio of the biofilm-attached Vallisneria and / or small stones to the denitrification culture medium is 1:1.5 g / mL.

[0044] Example 3

[0045] The method is the same as Example 1, except that: in step (1), the dark treatment is performed for 60 hours; in step (2), the mass volume ratio of the biofilm-attached Vallisneria and / or small stones to the denitrification culture medium is 1:2.5 g / mL.

[0046] The description of the above embodiments is only used to help understand the technical solution and core ideas of the present invention. It should be pointed out that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting N2O emissions from biofilms attached to submerged plants and / or non-submerged plants in freshwater lakes, characterized in that: The steps include: (1) Submerged plants and / or non-submerged plant carriers with biofilms collected from freshwater lakes were placed in ultrapure water for dark treatment; (2) placing the submerged plants and / or non-submerged plant carriers with biofilm attached after dark treatment in a serum bottle containing denitrification culture medium, sealing the bottle, and purging the gas in the serum bottle with N2 to remove N2O and O2 in the headspace; (3) Acetylene was added to the acetylene inhibitor group to a final volume fraction of 10% in the serum bottle, while no acetylene was added to the blank group; (4) The potential net N2O emission rate, potential N2O reduction rate, and potential denitrification rate of the biofilm were investigated using the above inhibitor group and blank group. The calculation formula is as follows: The N2O cumulative concentration is obtained by the following steps: taking out the gas in the inhibitor group and the blank group at 0-4 hours and detecting the N2O concentration.

2. The method according to claim 1, characterized in that In step (1), the dark treatment is performed for 24-60 hours.

3. The method according to claim 1, characterized in that The mass volume ratio of the submerged plant and / or non-submerged plant carrier to the denitrification culture solution in step (2) is 1:1.5-2.5 g / mL.

4. The method according to claim 3, characterized in that The mass volume ratio of the submerged plant and / or non-submerged plant carrier to the denitrification culture solution in step (2) is 1:2 g / mL.

5. The method according to claim 1 or 3, characterized in that The composition ratio of the denitrification culture medium described in step (2) is: potassium nitrate 8mg-N / L, glucose 40mg-C / L, potassium dihydrogen phosphate 0.4mg-P / L and trace elements 1mL / L, the pH is adjusted to 7.5 with saturated sodium bicarbonate solution, and the balance is water; the composition ratio of the trace elements is: manganese chloride 1.86g / L, zinc sulfate 0.22g / L, sodium molybdate 0.39g / L, boric acid 2.86g / L and copper sulfate 0.08g / L.

6. The method according to claim 1, characterized in that The N2O cumulative concentration described in step (4) is specifically obtained by the following steps: 10 mL of gas is taken out from the experimental bottle at 0, 0.5 h, 1 h, 2 h and 4 h respectively, and the N2O concentration is detected.

7. The method according to claim 6, characterized in that After taking out 10 mL of gas from the experimental bottle in step (4), 10 mL of N2 was injected at the same time to ensure the gas pressure balance in the experimental bottle.

8. Use of the method according to claim 1 in studying N2O emissions from biofilms attached to submerged plants and / or non-submerged plants in freshwater lakes.

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