CN111487365B - Method for in-situ determination of denitrification and anaerobic ammonia oxidation rates of deep-water reservoir sediments - Google Patents
Method for in-situ determination of denitrification and anaerobic ammonia oxidation rates of deep-water reservoir sediments Download PDFInfo
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Abstract
The invention discloses a method for in-situ determination of denitrification and anammox rates of deep-water reservoir sediments, which comprises the following steps: step one, preparing a sample; step two, carrying out an experimental test by using a sediment original position changing device; and step three, calculating the denitrification rate and the anaerobic ammonia oxidation rate according to the test result. The method can change sediment samples from the original position of the deep water reservoir, reduces the interference of the traditional sediment acquisition method, provides a method and basis for more accurately calculating the sediment denitrification and anaerobic ammonia oxidation rate of the reservoir, particularly the deep water reservoir, and has important significance for developing the denitrification research of the reservoir, particularly the deep water reservoir.
Description
Technical Field
The invention belongs to the technical field of environmental monitoring, relates to the field of water environment, and particularly relates to a method for in-situ determination of denitrification and anammox rates of deep-water reservoir sediments.
Background
Denitrification (Nitrogen loss) refers to the process by which organic or inorganic Nitrogen within a flow field is ultimately converted to a gas and released into the air, since only Nitrogen (N) is present at ambient temperature2) Since inert and harmless gases are involved, the nitrogen removal process by nitrogen generation is considered to be the most effective nitrogen load reduction process. The ecological system balance can be broken by the over-high concentration of the dissolved nitrogen in the ecological system, a series of ecological environment problems are generated, and how to adopt effective measures to reduce the nitrogen pollution load of rivers becomes a common problem in the world at present.
The currently approved dissolved nitrogen is directly converted into N2There are two main routes (denitrification process): (ii) Denitrification process (denitrifications): nitrate (NO-3) → nitrite (NO-2) → Nitric Oxide (NO) → nitrous oxide (N)2O)→N2(ii) a ② anaerobic ammonia oxidation process (Anammox): ammonia Nitrogen (NH)4 +)+NO-2(or NO-3)→N2。
The research on denitrification is a hot spot of international research at present, mainly sewage and farmland denitrification, and the paper about 'reservoir denitrification' accumulates more than 200 publications by 2018, and relatively few studies are internationally carried out. Although relatively more research works of 'denitrification' are carried out in China, the research works are mainly focused on the field of sewage treatment, the publications related to 'reservoir denitrification' are few, and 31 publications are accumulated on the network only in 2018. In fact, in China, reservoirs with different sizes are built in almost every watershed, the total number of the reservoirs is nearly 10 ten thousand, and the reservoirs (particularly step reservoir groups) inevitably have important influence on migration and transformation of nitrogen and phosphorus in the watershed due to the fact that the runoff process is completely changed, and high attention should be paid to the reservoirs. However, as can be seen from the above statistics, the "nitrogen circulation" and "denitrification efficiency" problems of the reservoir are not focused on worldwide, and the related research is much deficient in the many china in the reservoir.
At present, the problem of reservoir denitrification is getting more and more attention, and how to accurately and effectively measure the reservoir denitrification rate (mainly the denitrification rate and the anammox rate) is a key research problem.
There are several ways to determine the denitrification rate, i.e. direct determination of the product, e.g. N2Flux method, N2: ar ratio method, acetylene inhibition method, etc., and there are indirect estimation methods such as nitrogen mass balance method, natural stable nitrogen isotope mass balance method, N index method, etc,15N tracing and pairing technology and the like.15The N tracing and pairing technology has the advantage of synchronously measuring the denitrification rate and the anaerobic ammonia oxidation rate.15The N-tracer method is by adding to the study system15N-labelled nitrogen salts15NO-3Or15NO-2After a period of incubation, the product N is determined2Nitrogen isotope composition of (a)29N2、30N2) And then the denitrification rate and the anaerobic ammonia oxidation rate can be calculated simultaneously. During denitrification and anaerobic ammonia oxidation, N is generated2Are different in the reactants, and are thus obtained by addition15NO-3Or15NO-2The culture experiment of (1) can distinguish N produced by denitrification and anammox2. In the process of adding15NO-3In the culture experiment of (1) above,n produced by anammox2From NO2-and NH4 +The nitrogen atom of (1): 1 ratio pair formation, and denitrification is two NO-3Or NO-2The molecule is reduced via intermediates NO and N2O conversion and generation of N by isotope free pairing2. From product N2According to the isotope composition of the compound,14N14N、14N15n and15N15n will be respectively (1-F)2：2F(1-F)：F2Wherein F is in the culture system15The abundance of N. Produced of15N15N comes only from the contribution of denitrification, and14N15n is produced by both denitrification and anammox. Thus, using measured15N15N production can be calculated from the denitrification rate and the denitrification yield14N15N, by measurement14N15Total N production minus denitrification14N15With N production being effected by anaerobic ammonia oxidation14N15N, combined with anaerobic ammoxidation14N14N：14N15Production ratio of N (1-F): f, calculating the anaerobic ammonia oxidation rate.
The main problems in the prior art include:
the denitrification reaction is a complex process and is mainly influenced by natural conditions such as water flow, water temperature, nutritive salt, illumination and the like and biological factors such as death, sedimentation, aggregation, predation and the like of algae. The traditional sediment and overlying water collection is carried out by using a columnar mud collector developed by the Water environmental research institute of the Chinese Water conservancy and hydropower science institute, but the traditional sediment and overlying water collection is easy to disturb sediment and overlying water in the collection process and interfere with the accuracy of the experiment. In order to realize in-situ measurement of the denitrification and anammox rates of the deep water reservoir sediments, the key problem to be solved is how to reduce the disturbance on the samples as much as possible in the process of collecting the samples and reduce the influence of the traditional mud sampler on the samples.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the method for measuring the denitrification and anammox rates of the deep-water reservoir sediments in situ, the sediment samples can be changed from the deep-water reservoir in situ, the interference of the traditional sediment acquisition method is reduced, the method and the basis are provided for more accurately calculating the denitrification and anammox rates of the reservoirs, particularly the deep-water reservoir sediments, and the method has important significance for the development of reservoir denitrification researches, particularly the deep-water reservoir denitrification researches.
Therefore, the invention adopts the following technical scheme:
a method for in-situ determination of denitrification and anaerobic ammonia oxidation rates of deep water reservoir sediments comprises the following steps:
step one, preparing a sample;
step two, carrying out an experimental test by using a sediment original position changing device;
and step three, calculating the denitrification rate and the anaerobic ammonia oxidation rate according to the test result.
Preferably, the sediment original position replacing device comprises a steel wire rope, a heavy hammer starter, a rolling shaft, a heavy hammer, a rotary cover, a bracket, a balance weight, an anti-sinking plate, a culture tube protective sleeve, a culture tube and a rotary cover bolt; the bracket, the anti-sinking plate and the culture tube protective sleeve are welded together to form a device main body; a counterweight is added on the anti-sinking plate according to the water depth requirement to ensure that the device is in good contact with the sediment; the rolling shaft is positioned on the bracket, one end of the rolling shaft is connected with the heavy hammer through a rope, and the other end of the rolling shaft is connected with the bolt of the rotary cover; the weight is connected with the steel wire rope, and the weight starter is sleeved on the steel wire rope.
Preferably, the bracket and the sinking prevention plate are both made of steel plate materials; the culture tube is a hollow round tube made of polyethylene material and provided with openings at two ends, the diameter is 50mm, the thickness is 2mm, and the length is 500 mm; the culture tube protective sleeve is a hollow steel tube, the diameter of the culture tube protective sleeve is 60mm, the thickness of the culture tube protective sleeve is 5mm, and the length of the culture tube protective sleeve is 550 mm.
Preferably, in the step one, the sample preparation process is that a box type sampler is used for collecting the low-disturbance sediment sample and the overlying water, and the overlying water of the collection box is pumped to be dry and stored; then the culture tube with two open ends is vertically inserted into the sediment in the box type sampler, the valve at the bottom of the box type sampler is opened, the box type sampler is removed, the bottom end of the culture tube cover is sealed by a cover, and the sample is prepared.
Preferably, the specific process of step two is as follows:
2.1, collecting water covering the sediment by using a water sampler;
2.2, putting a sample to be reacted into a culture tube;
2.3, taking 20ml of redundant sample, mixing the redundant sample with overlying water, filling the mixture into a culture tube protective sleeve, then putting the prepared culture tube into the culture tube protective sleeve, ensuring that the experimental sample fills a gap between the culture tube and the culture tube protective sleeve, filling the overlying water into the culture tube protective sleeve, closing a rotary cover and inserting a rotary cover bolt;
2.4, slowly putting the whole sediment original position device into the water by using an electric winch until the sinking prevention plate contacts the surface layer of the sediment, putting a heavy hammer starter along the steel wire rope on the basis that the steel wire rope is not stressed any more, and opening a rotating cover;
and 2.5, after the reaction time is up, lifting the sediment original position replacing device out of the water surface, and testing relevant indexes.
Preferably, the specific process of step three is as follows: setting four same samples on each research point in different areas, and implanting the samples into the sediment according to the operation method of a sediment in-situ replacement device; taking out the culture tube after 10 days of reaction, collecting samples 1-6cm at a rate of 1 cm/layer, 6-20cm at a rate of 2 cm/layer, and 20cm later at a rate of 5 cm/layer, adding the samples into a sealed anaerobic sampling bottle purged with helium, and adding the samples into a calibrated anaerobic sampling bottle15NO-2Placing the mixture into a constant-temperature water bath kettle to vibrate for 40 minutes, then utilizing raw water to remove mixed gas after reaction for storage, and bringing the mixed gas into a laboratory for measurement by an isotope mass spectrometer14N15N and15N15and (4) N content.
Further, using measured values15N15Calculating the rate of denitrification and the production of denitrification by N production14N15N, by measurement14N15Total N production minus denitrification14N15Obtaining the anammox with N yieldProduced by14N15N, combined with anaerobic ammoxidation14N14N：14N15Production ratio of N (1-F): f, calculating the anaerobic ammonia oxidation rate.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method can change sediment samples from the original position of the deep water reservoir, reduces the interference of the traditional sediment acquisition method, provides a method and basis for more accurately calculating the sediment denitrification and anaerobic ammonia oxidation rates of the reservoir, particularly the deep water reservoir, and has important significance for developing the denitrification research of the reservoir, particularly the deep water reservoir.
(2) Simple structure, convenient use and accurate and reliable test result.
Drawings
FIG. 1 is a schematic structural diagram of a sediment in-situ conversion device used in the method for in-situ measuring the denitrification and anammox rates of deep water reservoir sediment.
FIG. 2 is a schematic view showing the detachment of a culture tube and a protective sheath for the culture tube.
Description of reference numerals: 1. a wire rope; 2. a weight dropper starter; 3. a roller; 4. a weight; 5. a rotating cover; 6. a support; 7. balancing weight; 8. a sinking prevention plate; 9. a culture tube protective sleeve; 10. a culture tube; 11. the cover latch is rotated.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments, which are provided for illustration only and are not to be construed as limiting the invention.
Examples
A method for in-situ measurement of denitrification and anammox rates of deep water reservoir sediments is mainly realized by a sediment in-situ device, the sediment in-situ device is shown as figure 1, wherein a schematic diagram of the culture tube and a culture tube protective sleeve in a split mode is shown as figure 2. A sediment original position replacing device comprises a steel wire rope 1, a heavy hammer starter 2, a rolling shaft 3, a heavy hammer 4, a rotary cover 5, a support 6, a balance weight 7, a sinking prevention plate 8, a culture tube protective sleeve 9, a culture tube 10 and a rotary cover bolt 11. The bracket 6, the anti-sinking plate 8 and the culture tube protecting sleeve 9 are welded together to form a device main body. The counter weight 7 can be added on the anti-sinking plate 8 according to the water depth requirement to ensure that the device is in good contact with the sediment; the roller 3 is positioned on the bracket 6, one end of the roller 3 is connected with the heavy hammer 4 through a rope, and the other end of the roller is connected with the bolt 11 of the rotary cover; the weight 4 is connected with the steel wire rope 1, and the weight starter 2 is sleeved on the steel wire rope 1.
And the bracket 6 and the sinking prevention plate 8 of the sediment original position device are both made of steel plate materials.
The culture tube 10 is a hollow round tube made of polyethylene material and having two open ends, the diameter of the tube is 50mm, the thickness of the tube is 2mm, and the length of the tube is 500 mm.
The culture tube protective sleeve 9 is a hollow steel tube, the diameter of the hollow steel tube is 60mm, the thickness of the hollow steel tube is 5mm, and the length of the hollow steel tube is 550 mm.
The specific method comprises the following steps: the sample preparation process comprises the steps of firstly collecting a low-disturbance sediment sample and overlying water by using a box type sampler, and draining and storing the overlying water in a collection box; then the culture tube 10 with two open ends is vertically inserted into the sediment in the box type sampler, the valve at the bottom of the box type sampler is opened, the box type sampler is removed, the bottom end of the culture tube 10 is sealed by a cover, and the sample is prepared.
The experimental test by using the sediment in-situ switching device comprises the following steps: firstly, collecting water covering the sediment by using a water sampler; secondly, putting a sample to be reacted into the culture tube 10; thirdly, taking 20ml of redundant samples, mixing the redundant samples with overlying water, filling the samples into a culture tube protective sleeve 9, then putting the prepared culture tube 10 into the culture tube protective sleeve 9, ensuring that the experimental samples fill the gap between the culture tube 10 and the culture tube protective sleeve 9, filling the overlying water into the culture tube protective sleeve 9, closing the rotary cover 5 and inserting the rotary cover bolt 11; fourthly, slowly putting the whole sediment original position device into the water by using an electric winch until the sinking prevention plate 8 contacts the surface layer of the sediment (taking the steel wire rope 1 as the standard for not bearing the force any more), putting the heavy hammer starter 2 along the steel wire rope 1, and opening the rotating cover 5; and fifthly, after the reaction time is reached, the sediment original position replacing device is lifted out of the water surface, and relevant indexes are tested.
Setting four same samples on each research point in different areas, and implanting the samples into the sediment according to the operation method of a sediment in-situ replacement device; after 10 days of reaction, taking out the culture tube 10, collecting samples 1-6cm at a rate of 1 cm/layer, 6-20cm at a rate of 2 cm/layer, and 20cm later at a rate of 5 cm/layer, adding the samples into a sealed anaerobic sampling bottle purged with helium, and adding the samples into a calibrated anaerobic sampling bottle15NO-2Shaking in constant temperature water bath for 40 min, storing with mixed gas obtained by raw water reaction, and measuring with isotope mass spectrometer (ThermoFisher DELTA V Advantage, USA)14N15N and15N15and (4) N content. From product N2According to the isotope composition of the compound,14N14N、14N15n and15N15n will be respectively (1-F)2：2F(1-F)：F2Wherein F is in the culture system15The abundance of N. Produced of15N15N comes only from the contribution of denitrification, and14N15n is produced by both denitrification and anammox. Thus, using measured15N15N production can be calculated from the denitrification rate and the denitrification yield14N15N, by measurement14N15Total N production minus denitrification14N15With N production being effected by anaerobic ammonia oxidation14N15N, combined with anaerobic ammoxidation14N14N：14N15Production ratio of N (1-F): f, calculating the anaerobic ammonia oxidation rate.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be covered thereby.
Claims (6)
1. A method for in-situ measurement of denitrification and anammox rates of deep water reservoir sediments is characterized by comprising the following steps:
step one, preparing a sample;
step two, carrying out an experimental test by using a sediment original position changing device;
calculating the denitrification rate and the anaerobic ammonia oxidation rate according to the test result;
the sediment original position replacing device comprises a steel wire rope (1), a heavy hammer starter (2), a rolling shaft (3), a heavy hammer (4), a rotary cover (5), a support (6), a balance weight (7), an anti-sinking plate (8), a culture tube protective sleeve (9), a culture tube (10) and a rotary cover bolt (11); the bracket (6), the sinking prevention plate (8) and the culture tube protective sleeve (9) are welded together to form a device main body; a counter weight (7) is added on the anti-sinking plate (8) according to the water depth requirement to ensure that the device is in good contact with the sediment; the rolling shaft (3) is positioned on the bracket (6), one end of the rolling shaft (3) is connected with the heavy hammer (4) through a rope, and the other end of the rolling shaft is connected with the rotating cover bolt (11); the weight (4) is connected with the steel wire rope (1), and the weight starter (2) is sleeved on the steel wire rope (1).
2. The method for in-situ measurement of the denitrification and anammox rates of deep water reservoir sediments according to claim 1, wherein the method comprises the following steps: the bracket (6) and the sinking-resisting plate (8) are both made of steel plate materials; the culture tube (10) is a hollow round tube made of polyethylene material and provided with openings at two ends, the diameter is 50mm, the thickness is 2mm, and the length is 500 mm; the culture tube protective sleeve (9) is a hollow steel tube, the diameter of the hollow steel tube is 60mm, the thickness of the hollow steel tube is 5mm, and the length of the hollow steel tube is 550 mm.
3. The method for in-situ measurement of the denitrification and anammox rates of deep water reservoir sediments as claimed in claim 1 or 2, wherein the method comprises the following steps: in the first step, the sample preparation process comprises the steps of firstly collecting a low-disturbance sediment sample and overlying water by using a box type sampler, and draining and storing the overlying water in a collection box; then the culture tube (10) with two open ends is vertically inserted into the sediment in the box type sampler, the valve at the bottom of the box type sampler is opened, the box type sampler is removed, the bottom end of the culture tube (10) is sealed by a cover, and the sample is prepared.
4. The method for in-situ measurement of the denitrification and anammox rate of the deep water reservoir sediments according to claim 1 or 2, wherein the specific process of the second step is as follows:
2.1, collecting water covering the sediment by using a water sampler;
2.2, putting a sample to be reacted into a culture tube (10);
2.3, taking 20ml of redundant sample, mixing the redundant sample with overlying water, filling the mixture into a culture tube protective sleeve (9), then putting the prepared culture tube (10) into the culture tube protective sleeve (9), ensuring that the experimental sample fills a gap between the culture tube (10) and the culture tube protective sleeve (9), filling the overlying water into the culture tube protective sleeve (9), closing a rotary cover (5) and inserting a rotary cover bolt (11);
2.4, slowly putting the whole sediment original position device into the water by using an electric winch until the sinking prevention plate (8) contacts the surface layer of the sediment, putting a heavy hammer starter (2) along the steel wire rope (1) on the basis that the steel wire rope (1) is not stressed any more, and opening a rotating cover (5);
and 2.5, after the reaction time is up, lifting the sediment original position replacing device out of the water surface, and testing relevant indexes.
5. The method for in-situ measurement of the denitrification and anammox rate of the deep water reservoir sediments according to claim 1 or 2, wherein the specific process of the third step is as follows: setting four same samples on each research point in different areas, and implanting the samples into the sediment according to the operation method of a sediment in-situ replacement device; after 10 days of reaction, taking out the culture tube (10), collecting samples 1-6cm at a rate of 1 cm/layer, 6-20cm at a rate of 2 cm/layer, and 20cm later at a rate of 5 cm/layer, adding the samples into a sealed anaerobic sampling bottle purged with helium, and adding the samples into a calibrated anaerobic sampling bottle15 NO2 -Placing the mixture into a constant-temperature water bath kettle to vibrate for 40 minutes, then utilizing raw water to remove mixed gas after reaction for storage, and bringing the mixed gas into a laboratory for measurement by an isotope mass spectrometerStator14N15N and15N15and (4) N content.
6. The method for in-situ measurement of the denitrification and anammox rates of deep water reservoir sediments as claimed in claim 5, wherein the method comprises the following steps: using actual measurements15N15Calculating the rate of denitrification and the production of denitrification by N production14N15N, by measurement14N15Total N production minus denitrification14N15With N production being effected by anaerobic ammonia oxidation14N15N, combined with anaerobic ammoxidation14N14N：14N15Production ratio of N (1-F): f, calculating the anaerobic ammonia oxidation rate, wherein F is in the culture system15The abundance of N.
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