Method and device for detecting water quality on-line biotoxicity through lactic acid bacteria PH inhibition method

Abstract

The invention discloses a water quality on-line biotoxicity detection method and a water quality on-line biotoxicity detection device through a lactic acid bacteria PH inhibition method. The detection method comprises the following steps: preparing a first water sample and a second water sample; acquiring initial PH voltage values of the first water sample and the second water sample; performing constant-temperature culture on the first water sample and the second water sample; respectively acquiring PH voltage values of the first water sample and the second water sample after culture; and performing biotoxicity analysis calculation on the to-be-detected water sample. According to the method disclosed by the invention, the size of harmful substances in water is determined by monitoring the pH change caused by lactic acid bacteria metabolite lactic acid; pH is detected based on a mature glass electrode electrochemical principle method, and the detection precision is not influenced by water turbidity; the detection method provided by the invention has high consistency with a photobacterium method in the prior art for detecting the biotoxicity, and meets the related technical requirements in the determination of the acute toxicity of the water quality.

Description

Technical Field

[0001] The present invention relates to the technical field of biological toxicity of water environment pollutants, and particularly to an on-line biological toxicity detection method and detection device for water quality by the lactic acid bacteria PH inhibition method. Background Art

[0002] Most of the current on-line biological toxicity analyzers on the market use luminescent bacteria (Vibrio fischeri or Photobacterium phosphoreum) for detection. The cultivation and long-term preservation of luminescent bacteria have relatively harsh conditions, and water turbidity will also affect the accuracy, etc.

[0003] In view of the above problems, those skilled in the art have begun to study the use of lactic acid bacteria for microbial toxicity monitoring. For example, the Chinese invention patent application with the application number 201910729523.2 discloses a sewage biological toxicity monitoring method, including the following steps: S1. Select 10 kinds of microorganisms, namely lactic acid bacteria, Escherichia coli, sulfur bacteria, nitrite bacteria, nitrite bacteria, denitrifying bacilli, actinomycetes, bacilli, rotifers and ciliates, as the characteristic communities of sewage treatment plants; S2. Select the influent COD concentration, influent total nitrogen concentration, influent water temperature, influent dissolved oxygen content, chromaticity, SS, sludge age and sludge concentration as environmental factors, and classify all sewage treatment plants according to the selected environmental factors; S3. Establish a reference group for each type of sewage treatment plant obtained in step S2; S4. When a certain sewage treatment plant in each type of sewage treatment plant operates unstably, measure its community structure, compare it with the reference group standard community structure of each type of sewage treatment plant, diagnose the sewage treatment plant, analyze the reasons from the microbial level, and provide a basis for the repair of the sewage treatment plant. Although this method uses lactic acid bacteria to evaluate the sewage treatment capacity of sewage treatment plants and monitor the effluent quality, the existing sewage biological toxicity monitoring method has high detection costs and complex detection methods. Summary of the Invention

[0004] The purpose of the present invention is to provide an on-line biological toxicity detection method and detection device for water quality by the lactic acid bacteria PH inhibition method to solve the problems raised in the background art.

[0005] To achieve the above purpose, the present invention provides an on-line biological toxicity detection method for water quality by the lactic acid bacteria PH inhibition method, including the following steps:

[0006] S1. Prepare a first water sample and a second water sample;

[0007] S2. Obtain the initial PH voltage values of the first water sample and the second water sample respectively;

[0008] S3. Incubate the first water sample and the second water sample at a constant temperature;

[0009] S4. Obtain the pH voltage values of the cultured first water sample and the second water sample respectively;

[0010] S5. Conduct biotoxicity analysis and calculation on the water sample to be tested.

[0011] Furthermore, the specific steps for preparing the water sample in step S1 are as follows:

[0012] S1.1. Clean the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add a certain amount of distilled water into the first culture tube and the second culture tube respectively to clean the first culture tube, the second culture tube, and the pH probes in the first culture tube and the second culture tube;

[0013] S1.2. Add the culture medium: The control system controls to add an equal amount of lactic acid bacteria culture medium into the first culture tube and the second culture tube respectively;

[0014] S1.3. The control system controls to add distilled water equal in amount to the added lactic acid bacteria culture medium into the first culture tube;

[0015] S1.4. The control system controls to add the water sample to be tested equal in amount to the added lactic acid bacteria culture medium into the second culture tube;

[0016] S1.5. Add the strains: The control system controls to add lactic acid bacteria strains equal in amount to the added lactic acid bacteria culture medium into the first culture tube and the second culture tube respectively. After mixing evenly, the first water sample and the second water sample are prepared.

[0017] Furthermore, in step S2, the control system reads the pH voltage values of the first pH sensor and the second pH sensor, so as to obtain the initial pH voltage value A1 of the first water sample and the initial pH voltage value B1 of the second water sample.

[0018] Furthermore, in step S3, keep the temperatures of the first culture tube and the second culture tube constant at 25 - 37 °C, and the constant temperature culture duration is 15 - 25 min.

[0019] Furthermore, in step S4, after the constant temperature culture ends, the control system reads the pH voltage values of the first pH sensor and the second pH sensor again, so as to obtain the pH voltage value A2 of the cultured first water sample and the pH voltage value B2 of the second water sample.

[0020] Furthermore, step S5 is specifically: Calculate the inhibition rate of the water sample to be tested using the following formula:

[0021]

[0022] Toxicity evaluation is performed on the water sample to be tested according to the calculation results; among them, if the CF is between 15% and 20%, it is slightly toxic; if the CF is between 20% and 35%, it is moderately toxic; if the CF is greater than 35%, it is highly toxic.

[0023] The present invention also provides an on-line biological toxicity detection device for water quality by the lactic acid bacteria PH inhibition method. The detection device includes a multi-way valve, a peristaltic pump, a first culture tube, a second culture tube, a metering unit, a positive sample bottle, a distilled water bottle, a water sample bottle to be tested, a waste liquid bucket, a lactic acid bacteria bottle, a culture medium bottle, a first PH sensor, a second PH sensor, a constant temperature module and a control system. The multi-way valve is respectively connected to the first culture tube, the second culture tube, the metering unit, the positive sample bottle, the distilled water bottle, the water sample bottle to be tested, the waste liquid bucket, the lactic acid bacteria bottle and the culture medium bottle. The first PH sensor and the second PH sensor are respectively arranged in the first culture tube and the second culture tube. The constant temperature module is used for constant temperature culture of the first culture tube and the second culture tube. The peristaltic pump is connected to the multi-way valve through the metering unit. The control system is respectively connected to the multi-way valve, the peristaltic pump, the metering unit, the first PH sensor, the second PH sensor and the constant temperature module.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The on-line biological toxicity detection method for water quality by the lactic acid bacteria PH inhibition method of the present invention cultivates lactic acid bacteria in a culture tube. As the lactic acid bacteria rapidly reproduce and grow, their metabolites will produce acidity. When lactic acid bacteria encounter harmful substances, it will affect the amount of their metabolites. By detecting the change of PH in the culture tube, the influence of poisons on the growth of lactic acid bacteria is judged. The method of the present invention determines the size of harmful substances in the water sample to be tested by monitoring the PH change caused by the metabolite lactic acid of lactic acid bacteria. Using the method of the present invention to detect biological toxicity has the characteristics of high detection accuracy, low use cost and simple maintenance.

[0026] (2) The on-line biological toxicity detection method for water quality by the lactic acid bacteria PH inhibition method of the present invention detects PH based on the mature glass electrode electrochemical principle method, and its detection accuracy is not affected by the turbidity of water quality. The method of the present invention for detecting biological toxicity has a high degree of consistency with the method of detecting biological toxicity by the luminescent bacteria method in the prior art, and meets the technical requirements in the determination of acute toxicity of water quality in GB / T15441-1995.

[0027] In addition to the purposes, features and advantages described above, the present invention has other purposes, features and advantages. The following will refer to the drawings to further describe the present invention in detail. Description of the Drawings

[0028] The accompanying drawings are used to provide a further understanding of the embodiments of the present invention and form a part of the specification. Together with the following specific embodiments, they are used to explain the embodiments of the present invention, but do not constitute a limitation to the embodiments of the present invention. In the accompanying drawings:

[0029] Figure 1 is a schematic structural diagram of an on-line biological toxicity detection device for water quality by the lactic acid bacteria PH inhibition method provided by the embodiment of the present invention;

[0030] Figure 2 is a flowchart of an on-line biological toxicity detection method for water quality by the lactic acid bacteria PH inhibition method provided by the embodiment of the present invention;

[0031] Among them, 1 - multi-way valve, 2 - peristaltic pump, 3 - first culture tube, 4 - second culture tube, 5 - metering unit, 6 - positive sample bottle, 7 - distilled water bottle, 8 - water sample bottle to be tested, 9 - waste liquid bucket, 10 - lactic acid bacteria bottle, 11 - culture medium bottle, 12 - first PH sensor, 13 - second PH sensor, 14 - constant temperature module. Specific Embodiments

[0032] The present invention will be described in detail below in conjunction with the various embodiments shown in the accompanying drawings. It should be noted, however, that these embodiments are not limitations to the present invention, and any equivalent transformation or substitution in terms of function, method, or structure made by those of ordinary skill in the art based on these embodiments shall fall within the protection scope of the present invention.

[0033] Please refer to Figure 1, an embodiment of the present invention also provides an on-line biological toxicity detection device for water quality by the lactic acid bacteria PH inhibition method, which includes a multi-way valve 1, a peristaltic pump 2, a first culture tube 3, a second culture tube 4, a metering unit 5, a positive sample bottle 6, a distilled water bottle 7, a water sample bottle to be tested 8, a waste liquid bucket 9, a lactic acid bacteria bottle 10, a culture medium bottle 11, a first PH sensor 12, a second PH sensor 13, a constant temperature module 14 and a control system. The inlets of the multi-way valve 1 are respectively connected to the first culture tube 3, the second culture tube 4, the metering unit 5, the positive sample bottle 6, the distilled water bottle 7, the water sample bottle to be tested 8, the waste liquid bucket 9, the lactic acid bacteria bottle 10 and the culture medium bottle 11. The inlets of the multi-way valve 1 are respectively connected to the first culture tube 3 and the second culture tube 4. The first PH sensor 12 and the second PH sensor 13 are respectively arranged in the first culture tube 3 and the second culture tube 4, and the constant temperature module 14 is used for carrying out constant temperature culture on the first culture tube 3 and the second culture tube 4. The peristaltic pump 2 is connected to the multi-way valve 1 through the metering unit 5. The control system is respectively connected to the multi-way valve 1, the peristaltic pump 2, the metering unit 5, the first PH sensor 12, the second PH sensor 13 and the constant temperature module 14. In this structural setting, the positive sample bottle 6, the distilled water bottle 7, the water sample bottle to be tested 8, the waste liquid bucket 9, the lactic acid bacteria bottle 10, and the culture medium bottle 11 are respectively used to contain positive test liquid, distilled water, water sample to be tested, waste liquid, lactic acid bacteria strains and lactic acid bacteria culture medium; the first PH sensor 12 and the second PH sensor 13 are respectively used to measure the PH voltage values of the liquids in the first culture tube 3 and the second culture tube 4.

[0034] Please refer to Figure 2 , this embodiment provides an on-line biological toxicity detection method for water quality by the lactic acid bacteria PH inhibition method. This detection method uses the above detection device for detection, and this detection method includes the following steps:

[0035] S1. Prepare a first water sample and a second water sample; the specific steps are as follows:

[0036] S1.1. Clean the culture tubes: The control system of the detection device controls the multi-way valve 1 and the peristaltic pump 2 to respectively add a certain amount of distilled water into the first culture tube 3 and the second culture tube 4 to clean the two culture tubes and the PH probes in the two culture tubes, so that the two culture tubes are kept clean.

[0037] S1.2. Add the culture medium: The control system controls the multi-way valve 1 and the peristaltic pump 2 to add 2 mL of lactic acid bacteria culture medium into the first culture tube 3 and the second culture tube 4.

[0038] S1.3. Add distilled water to the first culture tube (zero calibration): The control system controls the multi-way valve 1 and the peristaltic pump 2 to add 2 mL of distilled water into the first culture tube 3.

[0039] S1.4, Adding the water sample to be tested into the second culture tube (water sample test): The control system controls the multi-way valve 1 and the peristaltic pump 2 to add 2 mL of the water sample to be tested into the second culture tube 4.

[0040] S1.5, Adding bacterial strains: The control system controls the multi-way valve 1 and the peristaltic pump 2 to add 2 mL of lactic acid bacteria strains into the first culture tube 3 and the second culture tube 4 respectively. After mixing evenly, the first water sample and the second water sample are obtained.

[0041] S2, Obtaining the initial PH voltage values of the first water sample and the second water sample; The control system reads the PH voltage values of the first PH sensor 12 and the second PH sensor 13, so as to obtain the initial PH voltage value A1 of the water sample in the first culture tube 3 and the initial PH voltage value B1 of the water sample in the second culture tube 4.

[0042] S3, Conducting constant-temperature cultivation on the first water sample and the second water sample; The constant-temperature module 14 controls the temperatures of the first culture tube 3 and the second culture tube 4 to be constant at 35 °C, and the constant-temperature cultivation duration is 20 min, so that lactic acid bacteria metabolize to produce lactic acid.

[0043] S4, Obtaining the PH voltage values of the first water sample and the second water sample after cultivation respectively; After the constant-temperature cultivation ends, the control system reads and records the PH voltage value A2 of the first water sample after cultivation and the PH voltage value B2 of the second water sample after cultivation.

[0044] S5, Conducting biological toxicity analysis and calculation on the water sample to be tested: Use the following formula to calculate the inhibition rate CF of the water sample to be tested:

[0045]

[0046] Conducting toxicity evaluation according to the calculation results; Among them, if CF is between 15% and 20%, it is mild toxicity; if CF is between 20% and 35%, it is moderate toxicity; if CF is greater than 35%, it is severe toxicity.

[0047] S6, Cleaning the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add distilled water into the first culture tube and the second culture tube, and clean the culture tubes and the two PH probes in the culture tubes to keep the two culture tubes clean.

[0048] Positive test:

[0049] In the positive test of the online biological toxicity test of water quality by the lactic acid bacteria PH inhibition method in the embodiment of the present invention, that is, the positive control test, in the test solution preparation stage, the positive test solution is a Zn 2+ solution with a concentration of 7.5 mg / L to test the sensitivity of lactic acid bacteria to toxic substances. Under normal circumstances, the inhibition rate should be greater than 50%. The specific steps of the positive test are as follows:

[0050] Clean the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add a certain amount of distilled water into the first culture tube and the second culture tube, cleaning the two culture tubes and the pH probes in the two culture tubes to keep the two culture tubes clean.

[0051] Add the culture medium: The control system controls to add 2 mL of lactic acid bacteria culture medium into the first culture tube and the second culture tube respectively.

[0052] Add distilled water to the first culture tube (zero calibration): The control system controls to add 2 mL of distilled water into the first culture tube.

[0053] Add zinc sulfate standard solution to the second culture tube (positive test): The control system controls to add 2 mL of zinc sulfate standard solution (7.5 mg / L) into the second culture tube.

[0054] Add the bacterial strain: The control system controls to add 2 mL of lactic acid bacteria strain into the first culture tube and the second culture tube respectively, and mix evenly.

[0055] Read the signals of the culture tubes: The control system reads and records the pH voltage value A of the first culture tube 11 and the pH voltage value B of the second culture tube 11 .

[0056] Incubate at a constant temperature: Control the temperatures of the first culture tube and the second culture tube to be constant at 35 °C, and the incubation time at a constant temperature is 20 min to facilitate the lactic acid bacteria to metabolize and produce lactic acid.

[0057] Read the signals of the culture tubes: After 20 min, the control system reads and records the pH voltage value A of the first culture tube 12 and the pH voltage value B of the second culture tube 12 .

[0058] Clean the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add distilled water into the first culture tube and the second culture tube, cleaning the culture tubes and the two pH probes in the culture tubes to keep the two culture tubes clean.

[0059] In the positive test, use the above inhibition rate CF formula to calculate the inhibition rate of lactic acid bacteria in the water sample to be tested. At this time, the CF value should be greater than 50%.

[0060] Negative test:

[0061] The negative test of the on-line biological toxicity test of water quality by the lactic acid bacteria pH inhibition method in the embodiment of the present invention, that is, the negative control test, tests the stability of the lactic acid bacteria pH inhibition method for detecting biological toxicity. Under normal circumstances, the inhibition rate range should be -10% to 10%. The specific steps of the negative test are as follows:

[0062] Clean the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add a certain amount of distilled water into the first culture tube and the second culture tube, and clean the two culture tubes and the pH probes in the two culture tubes to keep the two culture tubes clean.

[0063] Add the culture medium: The control system controls to add 2 mL of lactic acid bacteria culture medium into the first culture tube and the second culture tube respectively.

[0064] Add distilled water to the first culture tube (zero calibration): The control system controls to add 2 mL of distilled water into the first culture tube.

[0065] Add distilled water to the second culture tube (zero calibration): The control system controls to add 2 mL of distilled water into the first culture tube.

[0066] Add the bacterial strain: The control system controls to add 2 mL of lactic acid bacteria strain into the first culture tube and the second culture tube respectively, and mix evenly.

[0067] Read the signal of the culture tube: The control system reads and records the pH voltage value A of the first culture tube 21 and the pH voltage value B of the second culture tube 21

[0068] Incubate at a constant temperature: Control the temperature of the first culture tube and the second culture tube to be constant at 35 °C, and the incubation time at a constant temperature is 20 min to facilitate the metabolism of lactic acid bacteria to produce lactic acid.

[0069] Read the signal of the culture tube: After 20 min, the control system reads and records the pH voltage value A of the first culture tube 22 and the pH voltage value B of the second culture tube 22 .

[0070] Clean the culture tubes: The control system controls the multi-way valve and the peristaltic pump to add a certain amount of distilled water into the first culture tube and the second culture tube, and clean the two culture tubes and the pH probes in the two culture tubes to keep the two culture tubes clean.

[0071] In the negative test, use the above inhibition rate CF formula to calculate the inhibition rate of lactic acid bacteria in the water sample to be tested. At this time, the CF value should be between -10% and 10%.

[0072] Test data

[0073] 1. Comparison of test data between the lactic acid bacteria pH inhibition method and the luminous bacteria method in the embodiments of the present invention

[0074] Take the water quality of a certain river at different times, and use the lactic acid bacteria in the embodiments of the present invention and the luminous bacteria method of the prior art to detect the biological toxicity respectively. The test results are as follows:

[0075] Table 1 Comparison of test data between the lactic acid bacteria pH inhibition method and the luminous bacteria method

[0076]

[0077] As can be seen from the data in Table 1, the test data of the lactic acid bacteria PH inhibition method in the embodiments of the present invention are highly consistent with those of the luminous bacteria method, further indicating the feasibility of detecting biological toxicity by the lactic acid bacteria PH inhibition method.

[0078] 2. Test data of positive tests:

[0079] Table 2 Test data of positive tests

[0080]

[0081] As can be seen from the data in Table 2, the CF value of the positive test is greater than 50%, and the test data are stable, meeting the requirements of the positive test.

[0082] 3. Test data of negative tests:

[0083] Table 3 Test data of negative tests

[0084]

[0085] As can be seen from the data in Table 3, the CF value of the negative test is between -10% and 10%, meeting the requirements of the negative test.

[0086] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An on-line biological toxicity detection method for water quality by lactic acid bacteria PH inhibition method, characterized in that, It includes the following steps: S1. Prepare the first water sample and the second water sample; S2. Obtain the initial PH voltage values of the first water sample and the second water sample respectively; S3. Incubate the first water sample and the second water sample at a constant temperature; S4. Obtain the PH voltage values of the first water sample and the second water sample after incubation respectively; S5. Conduct biotoxicity analysis and calculation on the water sample to be tested.

2. The detection method according to claim 1, characterized in that, The specific steps for preparing the water sample in step S1 are as follows: S1.

1. Clean the culture tubes: The control system controls the multi-way valve (1) and the peristaltic pump (2) to add a certain amount of distilled water into the first culture tube (3) and the second culture tube (4) respectively, and clean the first culture tube (3), the second culture tube (4) and the PH probes in the first culture tube (3) and the second culture tube (4); S1.

2. Add the culture medium: The control system controls to add an equal amount of lactic acid bacteria culture medium into the first culture tube (3) and the second culture tube (4) respectively; S1.

3. The control system controls to add distilled water equal to the added lactic acid bacteria culture medium into the first culture tube (3); S1.

4. The control system controls to add the water sample to be tested equal to the added lactic acid bacteria culture medium into the second culture tube (4); S1.

5. Add the bacterial strains: The control system controls to add lactic acid bacteria strains equal to the added lactic acid bacteria culture medium into the first culture tube (3) and the second culture tube (4) respectively, and after mixing evenly, the first water sample and the second water sample are prepared.

3. The detection method according to claim 2, wherein In step S2, the control system reads the PH voltage values of the first PH sensor (12) and the second PH sensor (13), so as to obtain the initial PH voltage value A1 of the first water sample and the initial PH voltage value B1 of the second water sample.

4. The detection method according to claim 3, wherein In step S3, keep the temperatures of the first culture tube (3) and the second culture tube (4) constant at 25 - 37 °C, and the constant temperature incubation duration is 15 - 25 min.

5. The detection method according to claim 4, wherein In step S4, after the constant temperature incubation ends, the control system reads the PH voltage values of the first PH sensor (12) and the second PH sensor (13) again, so as to obtain the PH voltage value A2 of the first water sample after incubation and the PH voltage value B2 of the second water sample.

6. The detection method according to claim 5, wherein Step S5 is specifically: Calculate the inhibition rate of the water sample to be tested by using the following formula: Conduct toxicity evaluation on the water sample to be tested according to the calculation result; among them, if CF is between 15% and 20%, it is mild toxicity; if CF is between 20% and 35%, it is moderate toxicity; if CF is greater than 35%, it is severe toxicity.

7. An on-line biological toxicity detection device for water quality by lactic acid bacteria PH inhibition method, characterized in that, The detection device includes a multi-way valve (1), a peristaltic pump (2), a first culture tube (3), a second culture tube (4), a metering unit (5), a positive sample bottle (6), a distilled water bottle (7), a water sample bottle to be tested (8), a waste liquid bucket (9), a lactic acid bacteria bottle (10), a culture medium bottle (11), a first pH sensor (12), a second pH sensor (13), a constant temperature module (14) and a control system. The multi-way valve (1) is respectively connected to the first culture tube (3), the second culture tube (4), the metering unit (5), the positive sample bottle (6), the distilled water bottle (7), the water sample bottle to be tested (8), the waste liquid bucket (9), the lactic acid bacteria bottle (10) and the culture medium bottle (11). The first pH sensor (12) and the second pH sensor (13) are respectively arranged in the first culture tube (3) and the second culture tube (4). The constant temperature module (14) is used for carrying out constant temperature culture on the first culture tube (3) and the second culture tube (4). The peristaltic pump (2) is connected to the multi-way valve (1) through the metering unit (5). The control system is respectively connected to the multi-way valve (1), the peristaltic pump (2), the metering unit (5), the first pH sensor (12), the second pH sensor (13) and the constant temperature module (14).

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