Application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium-oxidizing microorganisms

Abstract

The application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms belongs to the field of environmental nitrogen conversion determination. The present invention aims to solve the problem that the existing selective inhibitors of microbial activity will simultaneously inhibit the activity of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms, resulting in the difficulty in separately determining the nitrogen conversion rates of the two microorganisms, and the inhibitors pollute the environment. The application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms is used for determining the nitrogen conversion rates of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms.

Description

Technical Field

[0001] The invention belongs to the field of environmental nitrogen transformation determination. Background Art

[0002] Since the Industrial Revolution, nitrogen-containing substances have been widely used in life and production, resulting in a large amount of nitrogen being directly discharged into the natural environment, breaking the original nitrogen balance of the earth and causing a series of increasingly serious environmental problems. On the one hand, the large amount of nitrogen-containing sewage (wastewater) discharged from daily life and industrial production has caused a surge in nitrogen pollutants in the environment, causing eutrophication, algal blooms, ozone layer depletion and other problems. On the other hand, nitrogen, as a nutrient for agricultural crop planting, is heavily dependent on the application of a large amount of nitrogen fertilizer, but due to the low utilization rate of nitrogen fertilizer, a large amount of nitrogen fertilizer loss has further aggravated environmental nitrogen pollution. However, whether as the main accommodation environment for most nitrogen pollutants or as the main source of nutrients for crop planting, soil and water bodies are constantly converting a large amount of nitrogen into nitrogen gas and releasing it into the atmosphere through the action of various microorganisms. Therefore, identifying and evaluating the contribution of various microorganisms to nitrogen conversion can provide important information for promoting the removal of nitrogen pollutants or reducing the loss of nitrogen nutrients, and provide sufficient scientific basis for taking corresponding artificial measures. It is currently a research hotspot in the field of environmental nitrogen conversion.

[0003] Anaerobic ammonium oxidizing microorganisms are one of the main contributors to environmental nitrogen transformation losses discovered in recent years. They coexist with denitrifying microorganisms in anaerobic areas of various soils and water bodies. Anaerobic ammonium oxidizing microorganisms can convert ammonia nitrogen and nitrite into nitrogen gas and a small amount of nitrate (NH4 + -N:NO3 - -N=1:0.26, that is, anaerobic ammonium oxidizing microorganisms will produce 0.26 units of nitrate for every unit of ammonia nitrogen converted). However, due to the interweaving of nitrogen conversion substrates and products of different microorganisms, it is difficult to directly distinguish the contribution of different microorganisms to environmental nitrogen conversion. At present, a common method for studying the contribution of different microorganisms to environmental nitrogen conversion is to use selective inhibitors of microbial activity to inhibit other nitrogen conversion processes, thereby determining the nitrogen conversion rate of a certain microorganism. Another common method is to use isotope tracing to distinguish the nitrogen conversion rates of various microorganisms by using nitrogen labeled with different isotopes (N15 and N14) as substrates. Ultimately, the contribution of different microorganisms to environmental nitrogen conversion is determined by the nitrogen conversion rates of various microorganisms.

[0004] At present, some common selective inhibitors of microbial activity usually inhibit the activity of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms at the same time, resulting in the difficulty in measuring the nitrogen conversion rates of the two microorganisms separately, and there is a significant risk of secondary pollution to the environment. For example, inhibitors commonly used to inhibit the activity of denitrifying microorganisms (such as acetylene, etc.) will also significantly inhibit the activity of anaerobic ammonium oxidizing microorganisms. However, the substances currently reported to inhibit the activity of anaerobic ammonium oxidizing microorganisms either also inhibit the activity of denitrifying microorganisms or promote the activity of denitrifying microorganisms as substrates. It is difficult to effectively measure the nitrogen conversion contribution of the two microorganisms and are not suitable as selective inhibitors for inhibiting the activity of anaerobic ammonium oxidizing microorganisms. Therefore, active selective inhibitors for anaerobic ammonium oxidizing microorganisms have not yet been developed. The isotope tracer method uses nitrogen labeled with different isotopes as substrates to measure the nitrogen conversion rates of some microorganisms that are difficult to distinguish. However, this process usually needs to be carried out under the conditions of different combinations of labeled nitrogen substrates, and the nitrogen products obtained at the same time generally need to be sent to professional companies for determination. The operation process is cumbersome and complicated, and the technical requirements for operators are high. In addition, the cost of isotope tracer method is much higher than that of selective inhibitors of microbial activity and is not suitable for large-scale promotion and use. Summary of the invention

[0005] The present invention aims to solve the problem that the existing selective inhibitors of microbial activity will simultaneously inhibit the activity of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms, resulting in difficulty in separately measuring the nitrogen conversion rates of the two microorganisms and the inhibitors polluting the environment, and further provides the use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms.

[0006] The application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms is used to determine the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms. The specific determination method is as follows:

[0007] 1. Place the samples to be tested in a plurality of sealed containers respectively, introduce inert gas into the sealed containers and exhaust oxygen, then use a vacuum pump to exhaust the inert gas above the samples to be tested in the sealed containers, add ethane gas into some of the sealed containers, and add inert gas into other parts of the sealed containers, to obtain sealed containers supplemented with ethane gas and sealed containers supplemented with inert gas;

[0008] The sample to be tested contains both anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms; the volume ratio of ethane gas to the sample to be tested in the sealed container supplemented with ethane gas is ≥3, and the gas pressure of ethane gas in the sealed container is ≥1 atmosphere; the volume ratio of inert gas to the sample to be tested in the sealed container supplemented with inert gas is ≥3, and the gas pressure of inert gas in the sealed container is ≥1 atmosphere;

[0009] 2. Under the condition that the culture temperature is the same as the actual temperature of the sampling environment of the sample to be tested and the shaking table speed is 100rpm-150rpm, the closed container supplemented with ethane gas and the closed container supplemented with inert gas are cultured for 200min-600min, and the liquid in the closed container supplemented with ethane gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration and ammonia nitrogen concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate and nitrite removal rate under ethane conditions respectively; the liquid in the closed container supplemented with inert gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration, ammonia nitrogen concentration and nitrate concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate, nitrite removal rate and nitrate removal rate under inert gas conditions respectively;

[0010] 3. After the incubation is completed, the liquid in each closed container is taken to measure the volatile suspended solids concentration to obtain the volatile suspended solids concentration of the sample to be tested;

[0011] 4. Calculate the different nitrogen conversion rates in each anaerobic bottle to obtain the ammonia nitrogen conversion rate, nitrite conversion rate and nitrate conversion rate, respectively, in units of mg-N / (mg-VSS·d). The specific calculation method is:

[0012] Ammonia nitrogen conversion rate = ammonia nitrogen removal rate / volatile suspended solids concentration of the sample to be tested;

[0013] Nitrite conversion rate = nitrite removal rate / volatile suspended solids concentration of the sample to be tested;

[0014] Nitrate conversion rate = nitrate removal rate / volatile suspended solids concentration of the sample to be tested;

[0015] The unit of ammonia nitrogen removal rate is mg-N / (L·d), the unit of nitrite removal rate is mg-N / (L·d), the unit of nitrate removal rate is mg-N / (L·d), and the unit of volatile suspended solids concentration of the sample to be tested is mg-VSS / L;

[0016] 5. Calculate the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and the nitrogen conversion rate of denitrifying microorganisms in mg-N / (mg-VSS·d). The specific calculation method is:

[0017] The nitrate production rate of anaerobic ammonium-oxidizing microorganisms = (ammonia nitrogen conversion rate under inert gas conditions - ammonia nitrogen conversion rate under ethane conditions) × 0.26;

[0018] The nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms = (nitrite conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - (nitrite conversion rate under ethane conditions + ammonia nitrogen conversion rate under ethane conditions) - nitrate generation rate of anaerobic ammonium oxidizing microorganisms;

[0019] Nitrogen conversion rate of denitrifying microorganisms = (nitrite conversion rate under inert gas conditions + nitrate conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms.

[0020] The beneficial effects of the present invention are:

[0021] (1) The ethane used in the present invention can effectively inhibit the activity of anaerobic ammonium oxidizing microorganisms in the form of gas, and the degree of inhibition of the activity of anaerobic ammonium oxidizing microorganisms can reach 99.9±0.1%, while having no obvious effect on the activity of other microorganisms (such as denitrifying microorganisms), and the nitrogen conversion rates of the two microorganisms can be measured separately.

[0022] (2) The ethane used in the present invention is a non-toxic and harmless gas, which is easy to purchase and low in cost, and is used in small amounts. The residue after use can be directly discharged, and the secondary pollution to the environment is relatively small.

[0023] (3) The device used in the present invention has low requirements, is easy to purchase or can be made by yourself, and can be used repeatedly. There is no strict requirement for the volume of the device, and it is suitable for testing soil or water samples with a small sampling volume.

[0024] (4) The operation of the present invention is simple and has low technical requirements for operators.

[0025] The invention is used for application of ethane gas in inhibiting denitrification activity of anaerobic ammonia oxidizing microorganisms.

[0026] Instruction Manual

[0027] Figure 1 This is a graph showing the relationship between ethane concentration and anaerobic ammonium oxidizing microbial activity inhibition rate in Example 1. DETAILED DESCRIPTION

[0028] Specific implementation method 1: Application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms, which is used to measure the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms. The specific measurement method is as follows:

[0029] 1. Place the samples to be tested in a plurality of sealed containers respectively, introduce inert gas into the sealed containers and exhaust oxygen, then use a vacuum pump to exhaust the inert gas above the samples to be tested in the sealed containers, add ethane gas into some of the sealed containers, and add inert gas into other parts of the sealed containers, to obtain sealed containers supplemented with ethane gas and sealed containers supplemented with inert gas;

[0030] The sample to be tested contains both anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms; the volume ratio of ethane gas to the sample to be tested in the sealed container supplemented with ethane gas is ≥3, and the gas pressure of ethane gas in the sealed container is ≥1 atmosphere; the volume ratio of inert gas to the sample to be tested in the sealed container supplemented with inert gas is ≥3, and the gas pressure of inert gas in the sealed container is ≥1 atmosphere;

[0031] 2. Under the condition that the culture temperature is the same as the actual temperature of the sampling environment of the sample to be tested and the shaking table speed is 100rpm-150rpm, the closed container supplemented with ethane gas and the closed container supplemented with inert gas are cultured for 200min-600min, and the liquid in the closed container supplemented with ethane gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration and ammonia nitrogen concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate and nitrite removal rate under ethane conditions respectively; the liquid in the closed container supplemented with inert gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration, ammonia nitrogen concentration and nitrate concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate, nitrite removal rate and nitrate removal rate under inert gas conditions respectively;

[0032] 3. After the incubation is completed, the liquid in each closed container is taken to measure the volatile suspended solids concentration to obtain the volatile suspended solids concentration of the sample to be tested;

[0033] 4. Calculate the different nitrogen conversion rates in each anaerobic bottle to obtain the ammonia nitrogen conversion rate, nitrite conversion rate and nitrate conversion rate, respectively, in units of mg-N / (mg-VSS·d). The specific calculation method is:

[0034] Ammonia nitrogen conversion rate = ammonia nitrogen removal rate / volatile suspended solids concentration of the sample to be tested;

[0035] Nitrite conversion rate = nitrite removal rate / volatile suspended solids concentration of the sample to be tested;

[0036] Nitrate conversion rate = nitrate removal rate / volatile suspended solids concentration of the sample to be tested;

[0037] The unit of ammonia nitrogen removal rate is mg-N / (L·d), the unit of nitrite removal rate is mg-N / (L·d), the unit of nitrate removal rate is mg-N / (L·d), and the unit of volatile suspended solids concentration of the sample to be tested is mg-VSS / L;

[0038] 5. Calculate the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and the nitrogen conversion rate of denitrifying microorganisms in mg-N / (mg-VSS·d). The specific calculation method is:

[0039] The nitrate production rate of anaerobic ammonium-oxidizing microorganisms = (ammonia nitrogen conversion rate under inert gas conditions - ammonia nitrogen conversion rate under ethane conditions) × 0.26;

[0040] The nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms = (nitrite conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - (nitrite conversion rate under ethane conditions + ammonia nitrogen conversion rate under ethane conditions) - nitrate generation rate of anaerobic ammonium oxidizing microorganisms;

[0041] Nitrogen conversion rate of denitrifying microorganisms = (nitrite conversion rate under inert gas conditions + nitrate conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms.

[0042] This embodiment uses ethane (C2H6) to inhibit the nitrogen conversion activity of anaerobic ammonium oxidizing microorganisms, and the anaerobic ammonium oxidizing microorganisms belong to the family Anammoxaceae of the order Planctomycetales.

[0043] In this embodiment, the effective gas component for inhibiting the activity of anaerobic ammonium-oxidizing microorganisms is ethane, and the remaining gas components can be selected according to specific needs, or 100% ethane gas can be directly selected.

[0044] This embodiment selectively inhibits the activity of anaerobic ammonium oxidizing microorganisms and is effectively applied to determine and evaluate the contribution of microbial environmental nitrogen conversion. The method is simple to operate, low in cost, and uses non-toxic and harmless raw materials, and has broad application prospects in the field of environmental nitrogen conversion contribution evaluation.

[0045] Microorganisms that dominate nitrogen conversion in the environment include aerobic nitrifying microorganisms, denitrifying microorganisms, and anaerobic ammonium oxidizing microorganisms. Among them, because both denitrifying and anaerobic ammonium oxidizing microorganisms are anaerobic microorganisms, the nitrogen conversion contribution of aerobic nitrifying microorganisms can be directly distinguished by setting the experimental conditions to contain oxygen. The principle of this embodiment is based on the fact that the nitrogen substrates of anaerobic ammonium oxidizing microorganisms are ammonia nitrogen and nitrite. Ethane can inhibit the uptake of ammonia nitrogen by anaerobic ammonium oxidizing microorganisms and thus affect nitrite metabolism at the same time, thereby inhibiting the nitrogen conversion activity of anaerobic ammonium oxidizing. As a non-reactive gas, ethane will not have an effect on the activity of denitrifying microorganisms. This is because organic matter is a metabolic substrate for denitrifying microorganisms, which usually promotes the activity of denitrifying microorganisms. However, denitrifying microorganisms lack the enzymes necessary to metabolize ethane, an organic matter, and will not be affected by ethane. (Wang, S, Reid, MC. Kinetics of nitrous oxide mass transfer from porewater into root aerenchyma of wetland plants. J. Environ. Qual. 2020; 49: 1717–1729. https: / / doi.org / 10.1002 / jeq2.20162). Therefore, this embodiment can distinguish the nitrogen conversion contribution of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms.

[0046] The beneficial effects of this embodiment are:

[0047] (1) The ethane used in this embodiment can effectively inhibit the activity of anaerobic ammonium oxidizing microorganisms in the form of gas, and the degree of inhibition of the activity of anaerobic ammonium oxidizing microorganisms can reach 99.9±0.1%, while having no obvious effect on the activity of other microorganisms (such as denitrifying microorganisms), and the nitrogen conversion rates of the two microorganisms can be measured separately.

[0048] (2) The ethane used in this embodiment is a non-toxic and harmless gas that is easy to purchase and low in cost. The amount used is small, and the residue after use can be directly discharged, which has little secondary pollution to the environment.

[0049] (3) The device used in this embodiment has low requirements, is easy to purchase or can be made by yourself, and can be used repeatedly. There is no strict requirement for the volume of the device, and it is suitable for testing soil or water samples with a small sampling volume.

[0050] (4) The operation of this embodiment is simple and has low technical requirements for operators.

[0051] Specific implementation method 2: This implementation method is different from the specific implementation method 1 in that the sample to be tested in step 1 is soil or water. The rest is the same as the specific implementation method 1.

[0052] Specific implementation method 3: This implementation method is different from specific implementation method 1 or 2 in that when the sample to be tested is soil, the soil is diluted with water without nitrogen to obtain a mud-water mixture, and the mud-water mixture is used for culture determination. The rest is the same as specific implementation method 1 or 2.

[0053] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that when the sample to be tested is soil, the soil is diluted with water without nitrogen to obtain a mud-water mixture, and the mud-water mixture is used for culture determination. The rest is the same as specific embodiments 1 to 3.

[0054] Specific implementation mode 5: This implementation mode is different from specific implementation modes 1 to 4 in that the volume percentage of the inert gas in the inert gas described in step 1 is 95% to 100%, and the remaining gas component is carbon dioxide. The rest is the same as specific implementation modes 1 to 4.

[0055] Specific implementation example 6: This implementation example is different from specific implementation examples 1 to 5 in that the inert gas is helium, nitrogen or argon. The rest is the same as specific implementation examples 1 to 5.

[0056] Specific embodiment 7: This embodiment differs from Specific embodiments 1 to 6 in that the volume percentage of ethane in the ethane gas described in step 1 is 95% to 100%, and the remaining gas components are carbon dioxide. The rest is the same as Specific embodiments 1 to 6.

[0057] Specific embodiment 8: This embodiment differs from specific embodiments 1 to 7 in that in step 1, an inert gas is introduced into the sealed container and oxygen is exhausted, specifically, the sealed container is aerated with inert gas for 5 to 10 minutes to exhaust oxygen. The rest is the same as specific embodiments 1 to 7.

[0058] Specific implementation method 9: This implementation method is different from specific implementation methods 1 to 8 in that the sealed container in step 1 is an anaerobic bottle. The rest is the same as specific implementation methods 1 to 8.

[0059] Specific embodiment ten: This embodiment differs from specific embodiments one to nine in that: the ammonia nitrogen removal rate in step two is obtained by the following steps: plotting a graph with time as the abscissa and the ammonia nitrogen concentration sampled and measured at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the ammonia nitrogen removal rate, in units of mg-N / (L·d);

[0060] The nitrite removal rate in step 2 is specifically obtained by the following steps: plotting a graph with time as the abscissa and the nitrite concentration measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the nitrite removal rate, in units of mg-N / (L·d);

[0061] The nitrate removal rate in step 2 is obtained by the following steps: plotting a graph with time as the horizontal axis and the nitrate concentration measured by sampling at the corresponding time as the vertical axis, and fitting each point in the graph with a straight line. The slope of the straight line is the nitrate removal rate, in mg-N / (L·d). The rest is the same as in specific embodiments 1 to 9.

[0062] The following examples are used to verify the beneficial effects of the present invention:

[0063] Embodiment 1:

[0064] The inhibitory effect of ethane dosage on the nitrogen conversion activity of anaerobic ammonium oxidizing microorganisms was tested in the following steps:

[0065] 1. Place the samples to be tested in 8 sealed containers respectively, and the volume of the samples to be tested in each sealed container is 50 mL. Use a gas mixture of helium and carbon dioxide to aerate the sealed containers for 5 minutes to exhaust oxygen, cover them with rubber stoppers, and then use a vacuum pump to exhaust the gas above the samples to be tested in the sealed containers. Then, add 150 mL of eight different gases into the 8 sealed containers respectively to obtain gas-filled sealed containers;

[0066] The sample to be tested is a microbial mixture cultured in a laboratory bioreactor. According to biological sequencing, the abundance of anaerobic ammonia oxidizing microorganism Candidatus Kuenenia stuttgartiensis in the microbial mixture is 58.4%, the proportion of denitrifying microorganisms is very small, and the nitrogen conversion effect of denitrification is almost non-existent. The volatile suspended solids concentration of the microbial mixture is 3700±150 (mg-VSS)·L -1 , the total removal rate of ammonia nitrogen and nitrite is 542 mg-N / (L·d); the gas pressure in the closed container for replenishing gas is 1 atmosphere;

[0067] The eight gases are respectively 95% helium + 5% carbon dioxide gas mixture, 5% ethane + 90% helium + 5% carbon dioxide gas mixture, 10% ethane + 85% helium + 5% carbon dioxide gas mixture, 25% ethane + 70% helium + 5% carbon dioxide gas mixture, 50% ethane + 45% helium + 5% carbon dioxide gas mixture, 75% ethane + 20% helium + 5% carbon dioxide gas mixture, 95% ethane + 5% carbon dioxide gas mixture, 100% ethane; the percentages in the eight gases are all volume percentages;

[0068] 2. Inject nitrite and ammonia nitrogen into the closed container to make up the gas at one time, so that the initial concentration of nitrite and ammonia nitrogen is 15 mg·L -1 and 50mg·L -1 , and then placed in a shaking incubator at a temperature of 32°C and a rotation speed of 150rpm for 140min. During the culturing process, the liquid in the sealed container was taken and filtered every 20min. The nitrite concentration (spectrophotometry, GB 7493-87) and ammonia nitrogen concentration (Nessler's reagent spectrophotometry, HJ 535-2009) of the filtered liquid were measured to obtain the ammonia nitrogen removal rate and the nitrite removal rate respectively; the anaerobic bottle containing 95% helium + 5% carbon dioxide gas mixture was used as the blank control group, and the other anaerobic bottles containing ethane mixed gas were used as the experimental group. The experiment was set up with 3 repeated groups;

[0069] 3. Calculate the different nitrogen conversion rates in each anaerobic bottle to obtain the anaerobic ammonium oxidizing microbial activity of the experimental group and the anaerobic ammonium oxidizing microbial activity of the blank control group, respectively. The unit is mg-N / (mg-VSS·d). The specific calculation method is:

[0070] Anaerobic ammonium oxidizing microbial activity = (nitrite removal rate + ammonia nitrogen removal rate) / volatile suspended solids concentration of the sample to be tested;

[0071] The unit of ammonia nitrogen removal rate is mg-N / (L·d), the unit of nitrite removal rate is mg-N / (L·d), and the unit of volatile suspended solids concentration of the sample to be tested is mg-VSS / L;

[0072] 4. Calculation of the inhibition rate of anaerobic ammonium-oxidizing microorganism activity in each anaerobic bottle, in %, and the specific calculation method is:

[0073] Inhibition rate = [1-(anaerobic ammonium oxidizing microbial activity in the experimental group / anaerobic ammonium oxidizing microbial activity in the blank control group)] × 100%.

[0074] The volume percentage of helium in the gas mixture of helium and carbon dioxide described in step 1 is 95%;

[0075] The sealed container described in step 1 is a 200 mL anaerobic bottle with a rubber stopper.

[0076] The ammonia nitrogen removal rate in step 2 is specifically obtained by the following steps: plotting a graph with time as the abscissa and the ammonia nitrogen concentration measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the ammonia nitrogen removal rate, in units of mg-N / (L·d);

[0077] The nitrite removal rate in step 2 is obtained by the following steps: plotting a graph with time as the horizontal axis and the nitrite concentration measured by sampling at the corresponding time as the vertical axis, and fitting each point in the graph with a straight line. The slope of the straight line is the nitrite removal rate, and the unit is mg-N / (L·d).

[0078] Since Example 1 is intended to verify the inhibitory effect of ethane dosage on the activity of anaerobic ammonium oxidizing microorganisms, only the removal rates of substrate nitrite and ammonia nitrogen are used to calculate the activity of anaerobic ammonium oxidizing microorganisms in this example.

[0079] The purpose of introducing 5% carbon dioxide gas in this embodiment is to provide a small amount of carbon source required for the growth of microorganisms; however, in actual applications, due to the short test cycle, the gas may not be supplied and will not affect the activity of the microorganisms.

[0080] Figure 1 The figure is a relationship diagram of ethane concentration-anaerobic ammonia oxidizing microorganism activity inhibition rate in Example 1. The data processing results are all expressed as (mean±SD). Through the inhibition experiment of different headspace gas ethane concentrations on the activity of anaerobic ammonia oxidizing microorganisms, it is found that the activity of anaerobic ammonia oxidizing microorganisms decreases with the increase of ethane concentration. Under the conditions that the ratio of the inoculation volume of the anaerobic ammonia oxidizing microorganism mixture to the gas volume is 3 and the gas pressure is 1 standard atmosphere, the activity inhibition rate of 95% ethane on anaerobic ammonia oxidizing microorganisms exceeds 97%, and the activity inhibition rate of 100% ethane on anaerobic ammonia oxidizing microorganisms reaches 99.9%. The results show that the ethane proposed in the present invention has a very good inhibitory effect on the activity of anaerobic ammonia oxidizing microorganisms and can be used as a selective inhibitor of the activity of anaerobic ammonia oxidizing microorganisms.

[0081] Embodiment 2:

[0082] The application of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms is used to determine the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms. The specific determination method is as follows:

[0083] 1. Place the samples to be tested in 6 sealed containers respectively, and the volume of the samples to be tested in each sealed container is 50 mL. Use helium to aerate the sealed containers for 5 minutes and exhaust oxygen, cover with rubber stoppers, and then use a vacuum pump to exhaust the helium above the samples to be tested in the sealed containers. Add 150 mL of ethane gas to 3 sealed containers, and add 150 mL of helium to the other 3 sealed containers, to obtain sealed containers filled with ethane gas and sealed containers filled with helium;

[0084] The sample to be tested contains both anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms; the gas pressure of ethane gas in the sealed container supplemented with ethane gas is 1 atmosphere; the gas pressure of helium gas in the sealed container supplemented with helium gas is 1 atmosphere;

[0085] 2. Under the condition that the culture temperature is the same as the actual measured temperature of the sampling environment of the sample to be tested, 32°C, and the shaking speed is 150rpm, the sealed container supplemented with ethane gas and the sealed container supplemented with helium are cultured for 300 minutes. During the culture process, the liquid in the sealed container supplemented with ethane gas is taken out and filtered every 30 minutes, and the nitrite concentration and ammonia nitrogen concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate and nitrite removal rate under ethane conditions respectively; during the culture process, the liquid in the sealed container supplemented with inert gas is taken out and filtered every 30 minutes, and the nitrite concentration, ammonia nitrogen concentration and nitrate concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate, nitrite removal rate and nitrate removal rate under inert gas conditions respectively;

[0086] 3. After the incubation, take 10 mL of liquid from each sealed container to determine the volatile suspended solids concentration (loss on ignition method, HJ 761-2015) to obtain the volatile suspended solids concentration of the sample to be tested;

[0087] 4. Calculate the different nitrogen conversion rates in each anaerobic bottle to obtain the ammonia nitrogen conversion rate, nitrite conversion rate and nitrate conversion rate, respectively, in units of mg-N / (mg-VSS·d). The specific calculation method is:

[0088] Ammonia nitrogen conversion rate = ammonia nitrogen removal rate / volatile suspended solids concentration of the sample to be tested;

[0089] Nitrite conversion rate = nitrite removal rate / volatile suspended solids concentration of the sample to be tested;

[0090] Nitrate conversion rate = nitrate removal rate / volatile suspended solids concentration of the sample to be tested;

[0091] The unit of ammonia nitrogen removal rate is mg-N / (L·d), the unit of nitrite removal rate is mg-N / (L·d), the unit of nitrate removal rate is mg-N / (L·d), and the unit of volatile suspended solids concentration of the sample to be tested is mg-VSS / L;

[0092] 5. Calculate the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and the nitrogen conversion rate of denitrifying microorganisms in mg-N / (mg-VSS·d). The specific calculation method is:

[0093] The nitrate production rate of anaerobic ammonium-oxidizing microorganisms = (ammonia nitrogen conversion rate under inert gas conditions - ammonia nitrogen conversion rate under ethane conditions) × 0.26;

[0094] The nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms = (nitrite conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - (nitrite conversion rate under ethane conditions + ammonia nitrogen conversion rate under ethane conditions) - nitrate generation rate of anaerobic ammonium oxidizing microorganisms;

[0095] Nitrogen conversion rate of denitrifying microorganisms = (nitrite conversion rate under inert gas conditions + nitrate conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms.

[0096] The sample to be tested in step 1 is soil, and the soil is diluted with water that does not contain nitrogen to obtain a mud-water mixture, and the mud-water mixture is used for culture determination.

[0097] The soil is a large piece of soil sample taken from a sampling environment to be measured, without destroying the bulk structure of the soil, and is transferred to a laboratory where testing can be carried out within 2 hours of the sampling time, and the soil bulk structure is crushed and dispersed to remove impurities.

[0098] The volume percentage of the helium gas in step 1 is 100%; the volume percentage of the ethane gas in step 1 is 100%.

[0099] The sealed container described in step 1 is a 200 mL anaerobic bottle with a rubber stopper.

[0100] The ammonia nitrogen removal rate in step 2 is specifically obtained by the following steps: plotting a graph with time as the abscissa and the ammonia nitrogen concentration (Nessler's reagent spectrophotometry, HJ 535-2009) measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the ammonia nitrogen removal rate, and the unit is mg-N / (L·d);

[0101] The nitrite removal rate in step 2 is specifically obtained by the following steps: plotting a graph with time as the abscissa and the nitrite concentration (spectrophotometric method, GB 7493-87) measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the nitrite removal rate, in units of mg-N / (L·d);

[0102] The nitrate removal rate in step 2 is specifically obtained by the following steps: plotting a graph with time as the abscissa and the nitrate concentration (ultraviolet spectrophotometry, HJ / T 346-2007) measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line. The slope of the straight line is the nitrate removal rate, and the unit is mg-N / (L·d).

[0103] The data processing results of this embodiment are all expressed as mean ± SE. Therefore, the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms can be obtained (anaerobic conditions only consider the nitrogen conversion of denitrifying microorganisms and anaerobic ammonium oxidizing microorganisms), and then, according to the nitrogen conversion rate of the two microorganisms, the respective contributions of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms to the removal of nitrogen pollutants or the loss of nitrogen nutrients can be obtained, thereby providing sufficient scientific basis for taking corresponding artificial measures.

Claims

1. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms is characterized by It is used to determine the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms. The specific determination method is as follows:

1. Place the samples to be tested in a plurality of sealed containers respectively, introduce inert gas into the sealed containers and exhaust oxygen, then use a vacuum pump to exhaust the inert gas above the samples to be tested in the sealed containers, add ethane gas into some of the sealed containers, and add inert gas into other parts of the sealed containers, to obtain sealed containers supplemented with ethane gas and sealed containers supplemented with inert gas; The sample to be tested contains both anaerobic ammonium oxidizing microorganisms and denitrifying microorganisms; the volume ratio of ethane gas to the sample to be tested in the sealed container supplemented with ethane gas is ≥3, and the gas pressure of ethane gas in the sealed container is ≥1 atmosphere; the volume ratio of inert gas to the sample to be tested in the sealed container supplemented with inert gas is ≥3, and the gas pressure of inert gas in the sealed container is ≥1 atmosphere; The sample to be tested is soil or water; the sealed container is an anaerobic bottle; 2. Under the condition that the culture temperature is the same as the actual temperature of the sampling environment of the sample to be tested and the shaking table speed is 100rpm-150rpm, the closed container supplemented with ethane gas and the closed container supplemented with inert gas are cultured for 200min-600min, and the liquid in the closed container supplemented with ethane gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration and ammonia nitrogen concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate and nitrite removal rate under ethane conditions respectively; the liquid in the closed container supplemented with inert gas is taken and filtered every 20min-60min during the culture process, and the nitrite concentration, ammonia nitrogen concentration and nitrate concentration of the filtered liquid are measured to obtain the ammonia nitrogen removal rate, nitrite removal rate and nitrate removal rate under inert gas conditions respectively; The ammonia nitrogen removal rate is specifically obtained by the following steps: plotting a graph with time as the abscissa and the ammonia nitrogen concentration measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the ammonia nitrogen removal rate, in units of mg-N / (L·d); The nitrite removal rate is specifically obtained by the following steps: plotting a graph with time as the abscissa and the nitrite concentration measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the nitrite removal rate, in units of mg-N / (L·d); The nitrate removal rate is specifically obtained by the following steps: plotting a graph with time as the abscissa and the nitrate concentration measured by sampling at the corresponding time as the ordinate, and fitting each point in the graph with a straight line, the slope of the straight line is the nitrate removal rate, in units of mg-N / (L·d); 3. After the incubation is completed, the liquid in each closed container is taken to measure the volatile suspended solids concentration to obtain the volatile suspended solids concentration of the sample to be tested; 4. Calculate the different nitrogen conversion rates in each anaerobic bottle to obtain the ammonia nitrogen conversion rate, nitrite conversion rate and nitrate conversion rate, respectively, in units of mg-N / (mg-VSS·d). The specific calculation method is: Ammonia nitrogen conversion rate = ammonia nitrogen removal rate / volatile suspended solids concentration of the sample to be tested; Nitrite conversion rate = nitrite removal rate / volatile suspended solids concentration of the sample to be tested; Nitrate conversion rate = nitrate removal rate / volatile suspended solids concentration of the sample to be tested; The unit of ammonia nitrogen removal rate is mg-N / (L·d), the unit of nitrite removal rate is mg-N / (L·d), the unit of nitrate removal rate is mg-N / (L·d), and the unit of volatile suspended solids concentration of the sample to be tested is mg-VSS / L; 5. Calculate the nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms and the nitrogen conversion rate of denitrifying microorganisms in mg-N / (mg-VSS·d). The specific calculation method is: The nitrate production rate of anaerobic ammonium-oxidizing microorganisms = (ammonia nitrogen conversion rate under inert gas conditions - ammonia nitrogen conversion rate under ethane conditions) × 0.26; The nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms = (nitrite conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - (nitrite conversion rate under ethane conditions + ammonia nitrogen conversion rate under ethane conditions) - nitrate generation rate of anaerobic ammonium oxidizing microorganisms; Nitrogen conversion rate of denitrifying microorganisms = (nitrite conversion rate under inert gas conditions + nitrate conversion rate under inert gas conditions + ammonia nitrogen conversion rate under inert gas conditions) - nitrogen conversion rate of anaerobic ammonium oxidizing microorganisms.

2. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 1, characterized in that In step 1, when the sample to be tested is soil, the soil is diluted with water that does not contain nitrogen to obtain a mud-water mixture, and the mud-water mixture is used for culture determination.

3. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 1, characterized in that The soil described in step 1 is a large soil sample taken from the sampling environment to be measured, without destroying the bulk structure of the soil, and transferred to a laboratory where testing can be carried out within 2 hours of the sampling time, and the soil bulk structure is crushed and dispersed to remove impurities.

4. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 1, characterized in that The volume percentage of the inert gas in the inert gas described in step 1 is 95% to 100%, and the remaining gas component is carbon dioxide.

5. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 4, characterized in that The inert gas is helium, nitrogen or argon.

6. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 1, characterized in that The volume percentage of ethane in the ethane gas described in step 1 is 95% to 100%, and the remaining gas component is carbon dioxide.

7. The use of ethane gas in inhibiting the denitrification activity of anaerobic ammonium oxidizing microorganisms according to claim 1, characterized in that In step 1, an inert gas is introduced into the sealed container and oxygen is exhausted. Specifically, the sealed container is aerated with the inert gas for 5 minutes to 10 minutes to exhaust oxygen.

Images