JP4161111B2 - How to monitor hazardous substances in environmental water - Google Patents

Description

  The present invention relates to a method for monitoring harmful chemical substances in water using, for example, a microbial sensor for environmental water such as river water and lakes and water in each process of water and sewage.

  Biosensors use biological functional materials such as enzymes and antibodies, and living organisms such as microorganisms and cells as molecular identification elements that recognize chemical substances to be measured in sample water. In combination with a membrane immobilized by chemical inclusion or covalent bonding to a transducer such as an electrochemical detector, the molecular identification signal of the biological material is converted into an electrical signal to measure chemical substances in the sample water. It is a sensor to perform.

  The biosensor is a method in which sample water is brought into contact with the immobilized membrane of the biomaterial, and a concentration change of a substance generated or consumed by a biochemical reaction caused thereby is detected by an electrical output such as current or voltage (in the present invention). In this case, it is converted into a change in sensor output) and measured.

  In general, a biosensor using microorganisms (referred to as a microorganism sensor in the present invention) applied to a water quality measuring instrument measures the oxygen consumption state of microorganisms in an immobilized membrane with a dissolved oxygen electrode. In order to maintain the number and activity of microorganisms in the immobilized membrane (hereinafter referred to as “activity”) stably for as long as possible, the optimum temperature condition and optimum pH condition of the microorganism are maintained. In addition, measurement is performed using a buffer solution containing trace nutrients necessary for growth.

  For example, in the following Patent Document 1, a. A flow cell that holds an immobilized microbial membrane immobilizing nitrite-producing bacteria and that has two liquid flow paths in contact with each surface of the immobilized microbial membrane, and a gas permeable membrane on one surface of the immobilized microbial membrane. A microbial sensor in combination with a dissolved oxygen electrode brought into contact therewith, b. A circulation system for circulating a buffer solution containing ammonia nitrogen as a substrate of the nitrite-producing bacteria at a predetermined concentration on the one surface of the immobilized microorganism membrane; c. A liquid supply system that switches between sample water, the standard solution for calibrating the microorganism sensor, and washing water, saturates the dissolved oxygen amount of the sample water and the standard solution, and sends the solution to the other surface of the immobilized microorganism membrane D. There is disclosed a toxic substance detection apparatus comprising an arithmetic / control circuit for performing arithmetic processing on an output signal of the microorganism sensor and controlling operation.

  According to this poison detection device, a microorganism combining an immobilized microbial membrane that is immobilized with a porous polymer membrane and a dissolved oxygen electrode as a detector, with nitrite-producing bacteria that are extremely vulnerable to harmful substances immobilized alive. A sensor is configured, and a sample solution containing ammonia nitrogen, which is a substrate for nitrite-producing bacteria, and a constant concentration of trace components such as iron and magnesium, and sample water are mixed and flowed continuously. Therefore, if there are harmful substances in the sample water that inhibit the respiration of nitrite-purifying bacteria, the respiratory activity of the nitrite-producing bacteria is inhibited and the oxygen electrode output increases. The presence of harmful substances in water can be detected.

  However, when monitoring harmful substances contained in river water containing abundant nutrients using, for example, microbial sensor applied water quality measuring instrument as described above, immobilized microorganisms Since the activity of microorganisms in the membrane increases in a short period of time, as described below, there is a problem that the detection sensitivity of the microorganism sensor for harmful substances decreases.

  A mechanism of deterioration in detection sensitivity of the microbial sensor for harmful substances will be described with reference to FIG. FIG. 7 shows an example of the response of the above microorganism sensor to a cyan (hazardous substance) solution.

  In FIG. 7, the curve shown in (1) is an example of the sensor output when the immobilized microbial membrane is initially attached to the dissolved oxygen electrode, that is, when the microorganisms in the immobilized microbial membrane are at the optimum activity. As shown in the curve shown in (1), when the microorganisms in the immobilized microorganism membrane are at the optimum activity, the sensor output is normally about 5 mV at zero point calibration (I in FIG. 7), and the next sensor calibration (see FIG. 7) is near 0 mV, and then the process proceeds to continuous monitoring (III in FIG. 7).

  At this time, the sensor output changes in a state close to 0 mV. And, in FIG. 7, at the time of IVa and IVb, when a cyan solution, which is a toxic substance that exhibits a respiration inhibiting action on humans and organisms, is mixed in the sample water, respiration of microorganisms in the immobilized microbial membrane is inhibited. The consumption of dissolved oxygen in the sample water decreases, and the sensor output also increases according to the cyan concentration (0.05 mg / L for IVa and 0.2 mg / L for IVb). As a result, it is possible to detect that a harmful substance is mixed and its degree.

  However, if this microbial sensor is used to continuously measure polluted river water in a large city for about one week, the sensor output of the microbial sensor will be as shown by the solid line in FIG. The output is 0 mV. Further, contamination of harmful substances cannot be detected at a cyan solution concentration of 0.05 mg / L (IVa), and sensitivity is lowered even at a high concentration of IVb of 0.2 mg / L. This is because, as a result of the increased activity of microorganisms in the immobilized microbial membrane, oxygen is deficient in the immobilized microbial membrane, but the sensor output does not become 0 mV or less even in the oxygen deficient state. The sensor output is 0 mV. Therefore, for example, if a negative value of the sensor output can be taken, it is considered that the response to the harmful substance is a response as shown by a broken line in FIG.

  If this is expressed by the oxygen consumption rate of the microorganism sensor with respect to the harmful substance concentration, that is, the action response curve, it is as shown in FIG. Here, assuming that the detectable concentration of the microorganism sensor is a level at which the oxygen consumption rate is reduced by 10% in 20 minutes, the detection sensitivity for cyan is 0 at the beginning of the mounting of the immobilized microorganism membrane (curve (1) in FIG. 7). Although it was 05 mg / L, after continuous measurement of polluted river water for about one week (curve (2) in FIG. 7), the detection sensitivity deteriorates to 0.2 mg / L.

  In order to solve the above-described decrease in detection sensitivity of the microbial sensor for harmful substances, the following Patent Document 2 uses a microbial sensor composed of a membrane on which microorganisms are immobilized and a dissolved oxygen electrode. In a method for detecting harmful substances in water), there is disclosed a method for detecting harmful substances in environmental water, wherein the set temperature of the microorganism sensor is controlled by the value of the electrical output of the microorganism sensor.

According to this method, two threshold values are set for the sensor output value on the decrease side and the increase side of the output, and the two temperatures of the optimum temperature and the high temperature side temperature are set in the thermostatic chamber in which the microorganism sensor is installed. When the sensor output value reaches the threshold value, the temperature of the thermostatic chamber is controlled to suppress the growth or activity of microorganisms, thereby maintaining the detection sensitivity for harmful substances.
Japanese Patent Publication No. 7-85072 JP 2001-165893 A

  However, in the method disclosed in Patent Document 2, it is difficult to accurately grasp the degree of oxygen rate-determination, that is, the degree of increase in the activity of microorganisms in the immobilized microorganism, from the sensor output, and uniform temperature control. In the method, the temperature load may be excessively applied to the microorganism in the immobilized microorganism. As a result, it is understood that microorganisms are excessively damaged, the output of the sensor suddenly drops, and even if the temperature is lowered to the optimum temperature, it takes time to recover and may send a false alarm. It was.

  Therefore, an object of the present invention is to stably detect a harmful substance in environmental water using a microorganism sensor for a long period of time without deteriorating the detection sensitivity for the harmful substance even for various water quality samples. It is to provide a method capable of monitoring harmful substances.

In order to achieve the above object, one of the present invention provides at least two or more kinds of substances when detecting harmful substances in environmental water using a microorganism sensor composed of a membrane on which microorganisms are immobilized and a dissolved oxygen electrode . A substrate concentration-dependent response characteristic is obtained by measuring an output current value or an output voltage value of the microorganism sensor with respect to each substrate concentration using sensor calibration solutions having different substrate concentrations, and the substrate concentration-dependent response characteristic is determined according to the substrate concentration-dependent response characteristic. The present invention provides a method for monitoring harmful substances in environmental water characterized by controlling the activity of microorganisms by changing the set temperature of a microorganism sensor.

  In the said invention, when the said substrate concentration dependence response characteristic exceeds predetermined value, it is preferable to set the preset temperature of the said microorganisms sensor to temperature higher than the optimal growth temperature of this microorganism.

According to the above invention, when detecting a harmful substance in environmental water using a microorganism sensor composed of a membrane on which microorganisms are immobilized and a dissolved oxygen electrode, at least two types of sensor calibration solutions having different substrate concentrations By measuring the output current value or output voltage value of the microorganism sensor with respect to each substrate concentration and determining the substrate concentration-dependent response characteristics, it is possible to accurately grasp the activity of the immobilized microorganism. Then, by changing the set temperature of the microorganism sensor according to the substrate concentration dependent response characteristic, the set temperature of the microorganism sensor can be changed more accurately according to the activity of the microorganism, and excessive damage is given to the microorganism. The activity of microorganisms can be controlled without any problems. As a result, toxic substances can be monitored stably over a long period of time without reducing the detection sensitivity for toxic substances even in various water quality samples.

Another aspect of the present invention provides a linear relationship between the substrate concentration and the sensor output when detecting harmful substances in environmental water using a microorganism sensor comprising a membrane on which microorganisms are immobilized and a dissolved oxygen electrode. Using the sensor calibration solution with the substrate concentration in the relationship, the time until the output current value or the output voltage value of the microorganism sensor exceeds a predetermined value when changing from the steady operation state to the predetermined substrate concentration. Measuring the response speed at the predetermined substrate concentration by measuring, and controlling the activity of microorganisms by changing the set temperature of the microorganism sensor according to the response speed, harmful substances in environmental water The monitoring method is provided.

  In the said invention, when the said response speed exceeds a predetermined value, it is preferable to set the preset temperature of the said microorganisms sensor to temperature higher than the optimal growth temperature of this microorganism.

According to the above invention, when detecting harmful substances in environmental water using a microorganism sensor composed of a membrane on which microorganisms are immobilized and a dissolved oxygen electrode, the substrate concentration and the sensor output have a linear relationship. Using the sensor calibration solution of the concentration, when changing from a steady operation state to a predetermined substrate concentration, measure the time until the output current value or output voltage value of the microorganism sensor exceeds a predetermined value, By obtaining the response speed at a predetermined substrate concentration, the activity of the immobilized microorganism can be more easily grasped. By changing the set temperature of the microorganism sensor according to the response speed, the set temperature of the microorganism sensor can be changed more accurately according to the activity of the microorganism, and the microorganism can be obtained without excessive damage to the microorganism. The degree of activity can be controlled. As a result, toxic substances can be monitored stably over a long period of time without reducing the detection sensitivity for toxic substances even in various water quality samples.

  According to the present invention, the activity of microorganisms used in a microorganism sensor can be accurately grasped by obtaining the substrate concentration-dependent response characteristics of the microorganism sensor or the response speed at a predetermined substrate concentration. Then, by controlling the set temperature of the microorganism sensor stepwise according to the activity, the activity of the microorganism can be controlled without imposing an excessive temperature load on the microorganism. As a result, while preventing a sudden drop in sensor output caused by damaging microorganisms due to excessive temperature load, it suppresses a decrease in detection sensitivity to harmful chemical substances due to an increase in the activity of microorganisms used in the microorganism sensor be able to. Therefore, it is possible to stably monitor harmful substances over a long period of time without reducing the detection sensitivity for harmful substances even in various water quality samples.

  In the present invention, for example, a microorganism sensor having a structure as shown in FIG. 1 can be used as a microorganism sensor composed of a microorganism-immobilized membrane (hereinafter referred to as an immobilized microorganism membrane) and a dissolved oxygen electrode.

  As shown in FIG. 1, the microorganism sensor 1 includes a flow cell 20 having a flow path 20 a, an immobilized microorganism film 25, and a dissolved oxygen electrode 21 that measures the amount of dissolved oxygen in the test water. The immobilized microbial membrane 25 is placed on a stainless steel wire mesh 26 installed in the flow cell 20 so as to come into contact with the test water flowing in the front flow path 20a, and the dissolved oxygen electrode 21 is It is attached so as to be in close contact with the immobilized microorganism membrane 25. A negative electrode 23 is installed in the dissolved oxygen electrode 21 filled with the electrode solution, and the positive electrode 22 is installed so as to be in contact with one surface of the immobilized microorganism membrane 25 through the diaphragm 24. The diaphragm 24 is fixed to the dissolved oxygen electrode 21 main body by an O-ring 27 c and a washer 29.

  In the present invention, the microorganism used for the immobilized microorganism membrane is preferably a nitrifying bacterium, and particularly preferably an ammonia oxidizing bacterium. Examples of ammonia oxidizing bacteria include Nitrosomonas europaea ATCC25978.

  The immobilized microbial membrane can be prepared according to a known method. For example, a nitrifying bacterium is suspended in an aqueous sodium alginate solution, and this suspension is dropped on a porous cellulose membrane, and then another cellulose sheet. It can be produced by sandwiching the membrane and immobilizing the cells by gelling sodium alginate with an aqueous calcium chloride solution.

  One of the methods of the present invention is applied when a harmful substance in environmental water is detected using the microorganism sensor having the above-described configuration, and the microorganism bait in the immobilized microorganism membrane (hereinafter, A substrate concentration-dependent response characteristic is obtained by measuring a change in sensor output of the microorganism sensor when the substrate concentration is changed using a solution containing a substrate) (hereinafter referred to as a sensor calibration solution). The activity level of the microorganism is controlled by changing the set temperature of the microorganism sensor by grasping the height of the activity of the microorganism in the immobilized microorganism membrane from the dependence response characteristic.

  The substrate concentration-dependent response characteristic is obtained by measuring the sensor output difference of the microorganism sensor using at least two kinds of sensor calibration solutions having different substrate concentrations and plotting the sensor output difference with respect to each substrate concentration (horizontal axis: (Substrate concentration, vertical axis: sensor output difference).

  As shown in Example 1, the slope of the straight line correlates with the activity of microorganisms in the immobilized microorganism membrane, and the slope of the straight line is large when the activity of microorganisms in the immobilized microorganism membrane is high. When the activity is low, the slope becomes small. Therefore, if the slope of the straight line exceeds a predetermined value, that is, if the activity is too high, the set temperature of the microbial sensor is set to reduce the activity of the microorganism. When the slope of the straight line is within a predetermined value, that is, when the activity is optimal, the set temperature is adjusted to the optimal growth temperature.

  FIG. 6 shows a measurement example of the temperature characteristic of the relative activity of the microorganism. In this example, the optimum growth temperature is around 30 ° C., and the low temperature side and the high temperature side of this optimum growth temperature are growth inhibition temperatures. . However, in the present invention, the growth of microorganisms cannot be sufficiently inhibited at a temperature lower than the optimum temperature for growth, and the activity of microorganisms in the immobilized microbial membrane cannot be reduced eventually. As the inhibition temperature, it is preferable to employ a temperature higher than the optimum growth temperature.

  For example, in the case of a microbial sensor using an immobilized microbial membrane using ammonia-oxidizing bacteria, ammonia nitrogen is used as a substrate, and at least two or more different substrate concentrations (usually the ammonia nitrogen concentration is 0 to 10 mg / day). L at least one kind of L and at least one kind of 10 mg / L or more sensor calibration solution are used to measure the sensor output difference in each sensor calibration solution and plot the sensor output difference against each substrate concentration. The slope of the straight line obtained (substance concentration dependent response characteristics) is obtained.

  When the slope of the straight line is large (when the activity of microorganisms in the immobilized microbial membrane is high), specifically, when the slope of the straight line is 6 or more, the set temperature of the microorganism sensor is adjusted to that of ammonia oxidizing bacteria. The growth inhibition temperature is 35 to 45 ° C., and preferably adjusted within the range of +5 to 10 ° C. from the optimum growth temperature (28 to 30 ° C.). In addition, temperature adjustment is performed stepwise according to the inclination of the straight line, for example, 37 ° C. when the slope of the straight line is 6 to 6.5, and 40 ° C. when the straight line exceeds 6.5. It is preferable. In addition, when the slope of the straight line is small (when the activity of microorganisms in the immobilized microorganism membrane is optimal), specifically, when the slope of the straight line is less than 6, the set temperature of the microorganism sensor is set to ammonia. What is necessary is just to adjust to 28-30 degreeC which is the optimal growth temperature of oxidation bacteria.

  According to this method, the activity of microorganisms in the immobilized microorganism membrane can be accurately grasped, and the temperature can be finely adjusted according to this activity, so that an excessive temperature load is applied to the microorganisms. The activity of microorganisms can be controlled.

  Another method of the present invention is to detect the harmful substance in the environmental water using the microorganism sensor having the above-described configuration, when the microorganism is changed from a steady operation state to a predetermined substrate concentration. By measuring the time until the output current value or output voltage value of the sensor exceeds a predetermined value, the response speed at the predetermined substrate concentration is obtained, and the set temperature of the microorganism sensor is changed according to the response speed. Thus, the activity of the microorganism is controlled.

  In this method, using one sensor calibration solution having a predetermined substrate concentration in which the relationship between the substrate concentration and the sensor output is linear, the time until the sensor output for the sensor calibration solution exceeds a predetermined value ( Response rate) is measured, and when this response rate exceeds a predetermined value, that is, when the activity of microorganisms in the immobilized microorganism membrane is high, in order to reduce the activity, set the temperature of the microorganism sensor in the same manner as above. When the response speed is within a predetermined value, that is, when the activity is optimum, the set temperature is adjusted to the optimum growth temperature.

  For example, in the case of a microbial sensor using an immobilized microbial membrane using ammonia-oxidizing bacteria, ammonia nitrogen is used as a substrate, and the sensor calibration solution (concentration range in which the ammonia nitrogen concentration and the sensor output are linearly related ( Usually, one ammonia nitrogen concentration of 1 to 5 mg / L is prepared, and when the sensor calibration solution starts flowing from a steady operating state, the sensor output is a predetermined value (for example, 90% response output equivalent). Time) (response speed) is measured. When the response speed to the sensor calibration solution is fast (when the activity of microorganisms in the immobilized microorganism membrane is high), specifically when the response speed is within 15 minutes, the set temperature of the microorganism sensor is set to ammonia. It is 35-45 degreeC which is the growth inhibition temperature of oxidation bacteria, Preferably, it adjusts in the range of + 5-10 degreeC from the optimal growth temperature (28-30 degreeC). In addition, it is preferable that temperature adjustment is adjusted in steps according to the inclination of the straight line. In addition, when the rate is slow (when the activity of microorganisms in the immobilized microorganism membrane is optimal), specifically when the response rate is 15 minutes or more, the temperature set for the microorganism sensor is set to grow ammonia-oxidizing bacteria. What is necessary is just to adjust to 28-30 degreeC which is the optimal temperature. Note that the substrate concentration in the steady operation state is set to, for example, an ammoniacal nitrogen concentration of 10 mg / L.

  In this method, since the activity of microorganisms in the immobilized microorganism membrane can be measured with one type of sensor calibration solution, the activity can be grasped more easily than the method described above.

  In the method of the present invention, the activity of microorganisms in the immobilized microbial membrane is measured at least once a day (usually about once a day) during the monitoring period, and an appropriate microorganism sensor according to the state of the activity. It is preferable to adjust the set temperature.

  FIG. 2 is a flow chart showing an example of a microbial sensor applied water quality measuring device for carrying out the method of the present invention. Hereinafter, the method of the present invention will be described with reference to FIG.

  This microbial sensor applied water quality measuring instrument has a microbial sensor 1 installed as shown in FIG. 1 in a thermostatic chamber 2 adjusted to a predetermined temperature (usually the optimum temperature for growth of microorganisms of the microbial sensor). It comprises a measuring unit 5, a sensor output display unit 6, a control unit 7, a recorder 8, and a liquid feeding unit 14 for feeding water for testing and sensor calibration, and further includes an air pump 12 and a pressure sensor 13. It is possible to aerate the test water.

  The test water sent by the liquid feed pump 11a is aerated, adjusted to a predetermined temperature by the heat exchanger 3, and then comes into contact with the microorganism sensor 1 to be drained.

  And when measuring the activity of the microorganisms in the fixed microorganism membrane of a microorganism sensor, operation is performed as follows.

  First, taking advantage of the fact that the microorganisms in the immobilized microbial membrane cannot act unless a substrate is given and dissolved oxygen is not consumed, the electromagnetic valve 9a is closed to stop feeding the sample water, The valve 9b and the electromagnetic valve 9d are opened, and the buffer solution A and pure water containing no harmful substances and substrates are fed by the pump 11b and the pump 11a, respectively, and the stabilized sensor output of the microorganism sensor 1 is stored in the device. And perform zero point calibration. The sensor output in this case is a value corresponding to the dissolved oxygen concentration in water.

  Next, a substrate having a known concentration that does not contain harmful substances is given to the microorganism in the immobilized microorganism membrane, and the dissolved oxygen concentration is measured when the microorganism normally operates. In the present invention, at least two types of sensor calibration solutions having different substrate concentrations are flowed, and the sensor output at each substrate concentration is stored in the apparatus. Specifically, the electromagnetic valve 9d is closed, the electromagnetic valve 9e is opened, and a sensor calibration solution (buffer B) containing a substrate with a predetermined concentration and pure water are fed to stabilize the output of the microorganism sensor 1. The sensor output after conversion is stored in the device.

  Thereafter, the electromagnetic valve 9e is closed, the electromagnetic valve 9f is opened, and a sensor calibration solution (buffer C) having a substrate concentration different from that of the sensor calibration solution (buffer B) and pure water are sent. The sensor output after stabilizing the output of the microorganism sensor 1 is stored in the apparatus. The sensor output in these cases is a value corresponding to the remaining dissolved oxygen concentration consumed by the microorganism activity of the microorganism sensor. As the buffer solutions A to C, a buffer solution having a buffering ability is used near the pH at which microorganisms of the microorganism sensor function stably. For example, in the case of a microorganism sensor using ammonia-oxidizing bacteria, pH 8 to A phosphate buffer solution having a buffer capacity around 9 can be used.

  Then, the slope of the straight line obtained by plotting the sensor output difference at each substrate concentration (substrate concentration-dependent response characteristic) is obtained from each measurement result, and the activity of the microorganism of the microorganism sensor is determined according to the above criteria. The temperature of the thermostat 2 is adjusted according to the activity. In a normal state, the thermostatic chamber 2 is adjusted to the optimum temperature for growth of microorganisms in the microorganism sensor. When the activity is too high, the temperature is higher than the optimum temperature for growth (preferably +5 to +5). It may be adjusted stepwise within the range of 10 ° C. Thereby, the activity can be maintained in a preferable state without giving an excessive temperature load to the microorganisms of the microorganism sensor.

  In the above method, the activity of microorganisms in the immobilized microorganism membrane is measured using at least two types of sensor calibration solutions having different substrate concentrations (the buffer B or the buffer C). Alternatively, one sensor calibration solution having a predetermined substrate concentration in which the relationship between the substrate concentration and the sensor output has a linear relationship is used, and the response speed to the sensor calibration solution is measured.

  Then, after adjusting the temperature of the thermostatic chamber 2, the electromagnetic valve 9f and the electromagnetic valve 9b are closed, the electromagnetic valve 9a is opened, water is supplied, and monitoring is started.

  The following experiment was conducted using the microorganism sensor applied water quality measuring instrument having the configuration shown in FIG. As the immobilized microorganism membrane of the microorganism sensor 1, an immobilized microorganism membrane having Nitrosomonas europaea ATCC25978 immobilized thereon was used.

  In FIG. 2, the solenoid valve 9d and the solenoid valve 9b are opened, and for the first zero point calibration (first calibration), a phosphate buffer solution (pH 8 to 9) (buffer solution A) containing no ammonia nitrogen is added. Water was allowed to flow, and the stabilized current value of the microorganism sensor 1 was stored.

  Next, the solenoid valve 9d is closed, the solenoid valve 9e is opened, and a phosphate buffer solution (pH 8 to 9) (buffer solution B) containing 0 to 10 mg / L of ammonia nitrogen and pure water are flowed to stabilize the sensor output. The current value after conversion was stored.

  Further, the solenoid valve 9e is closed, the solenoid valve 9f is opened, and a phosphate buffer solution (pH 8 to 9) (buffer solution C) containing ammonia nitrogen at a concentration of 10 mg / L or more and pure water are flowed to stabilize the sensor output. The later voltage value was stored.

  In addition, the measurement as described above is performed for immobilized microbial membranes A to D (high activity: A> B> C> D) having different microbial activities, and each substrate of the immobilized microbial membranes A to D is measured. The sensor output difference in concentration was measured, and the sensor output difference in each ammonia concentration was plotted. The result is shown in FIG. FIG. 3 shows that the relationship between the ammonia concentration and the sensor output has a linear relationship within a certain ammonia concentration range.

  FIG. 4 shows the relationship between the substrate concentration-dependent response of each immobilized microbial membrane (straight line in FIG. 3) and the sensitivity (inhibition (%)) to harmful substances.

Note that inhibition in FIG 4 (%), the adverse cyan 0.05 mg / L as a substance ^- shed (KCN of CN as) was added water as test water, respiratory inhibition rate calculated by the following equation (1) means.
Respiration inhibition rate (%) = {(V _M −V ₂ ) / (V ₁ −V ₂ )} × 100 (1)
V ₁ : Sensor output of zero calibration V ₂ : Sensor output of feed liquid V _M : Sensor output of test water As a feed liquid, buffer solution is used for V ₁ measurement, and ammonia is used as a buffer solution for V ₂ measurement. liquid sex nitrogen was added to a 5 to 10 mg / L, further, as the for _{V M} measurements used was added to a ammonia nitrogen 1 to 5 mg / L.

  From FIG. 4, the greater the slope of the straight line (the activity of microorganisms in the immobilized microorganism membrane), the smaller the value of inhibition (%) by harmful substances, resulting in lower sensitivity of the microorganism sensor. I understand that. In FIG. 4, if the respiratory inhibition rate (%) is 10% or more, it is determined that the level is detectable.

  Using a sample water obtained by adding cyan as a harmful substance to polluted river water in a large city using the same microorganism sensor applied water quality measuring instrument as in Example 1 (the temperature of the thermostat 2 is initially adjusted to 30 ° C.) The change over time in the detection sensitivity (respiration inhibition rate (%)) for cyanide was measured for about 1 month. In addition, the respiratory inhibition rate (%) was calculated by the above formula (1) of Example 1.

  Specifically, water containing 0.05 mg / L or 0.2 mg / L of cyan as a harmful substance in river water is run as test water, and once a day, the microorganism sensor is subjected to the same method as in Example 1. The substrate concentration-dependent characteristics are measured to determine the activity of microorganisms in the immobilized microorganism membrane. When the slope of the straight line is less than 6, the set temperature of the thermostat 2 is kept at 30 ° C., and 6-6. In the case of 5, when it exceeded 37 degreeC and it exceeded 6.5 degreeC, it changed to 40 degreeC in steps, and monitoring was performed, controlling the activity of microorganisms. The result is shown in FIG. FIG. 5 shows that the sensitivity to cyan 0.05 mg / L can be maintained for one month or more even when nutrient-rich river water in a large city is used as a test water. If the respiratory inhibition rate (%) is 10% or more, it is determined that the level is detectable.

  Therefore, according to the method of the present invention, the sensor output is prevented from abruptly decreasing due to damage to the microorganisms of the microorganism sensor due to an excessive temperature load, and is stable over a long period of time without reducing the detection sensitivity to harmful substances. It can be seen that harmful substances can be monitored.

  The method of the present invention can maintain a high sensitivity for detection of harmful substances in the microbial sensor regardless of the quality of the test water. For example, the activity of the microorganisms used in the microbial sensor increases, The present invention can be suitably applied to monitoring of harmful substances targeting water rich in nutrients such as polluted river water in large cities where the detection sensitivity to harmful chemical substances tends to decrease in a short period of time.

It is a schematic diagram which shows an example of a structure of a microorganisms sensor. It is a flowchart which shows an example of a structure of a microorganisms sensor applied water quality measuring device. It is a figure explaining the substrate (ammonia) density | concentration dependence of a microorganism sensor. It is a figure explaining the relationship between the value (activity) of the inclination of a concentration dependence characteristic (linear approximation), and the sensitivity with respect to a harmful substance. It is a figure which shows the result of Example 2 of this invention. It is a figure explaining the growth temperature characteristic of microorganisms. It is a figure which shows the fall of the detection sensitivity with respect to a harmful substance. It is a figure which shows the relationship of the oxygen consumption rate of the microorganisms sensor with respect to the harmful | toxic substance density | concentration in FIG.

Explanation of symbols

1. 1. Microorganism sensor 2. Thermostatic bath Heat exchanger 4. 4. Two-way switching three-way valve Measurement unit 6. Display unit 7. Control unit 8. Recorders 9a-9h. Solenoid valve 10. Roller clamps 11a to 11b. Pump 12. Air pump 13. Pressure sensor 20. Flow cell 20a. Sample channel 21. Dissolved oxygen electrode 22. Positive electrode 23. Negative electrode 24. Diaphragm 25. Immobilized microbial membrane 26. Stainless steel wire mesh 27a-27c. O-rings 28a-28b. Lead wire 29. Washer

Claims (4)

When detecting harmful substances in environmental water using a microorganism sensor composed of a membrane with immobilized microorganisms and a dissolved oxygen electrode, each substrate concentration is measured using at least two types of sensor calibration solutions with different substrate concentrations. By measuring the output current value or the output voltage value of the microorganism sensor with respect to the substrate concentration-dependent response characteristics, and by changing the set temperature of the microorganism sensor according to the substrate concentration-dependent response characteristics, the activity of microorganisms A method for monitoring harmful substances in environmental water, characterized by controlling water.   The method for monitoring harmful substances in environmental water according to claim 1, wherein when the substrate concentration-dependent response characteristic exceeds a predetermined value, the set temperature of the microorganism sensor is set to a temperature higher than the optimum growth temperature of the microorganism. A sensor calibration solution with a substrate concentration that has a linear relationship between the substrate concentration and the sensor output when detecting harmful substances in environmental water using a microorganism sensor composed of a membrane with immobilized microorganisms and a dissolved oxygen electrode And measuring the time until the output current value or output voltage value of the microorganism sensor exceeds a predetermined value when changing from a steady operation state to a predetermined substrate concentration. A method for monitoring harmful substances in environmental water, characterized in that the activity rate of microorganisms is controlled by obtaining a response speed in the method and changing a set temperature of the microorganism sensor in accordance with the response speed.   4. The method for monitoring harmful substances in environmental water according to claim 3, wherein when the response speed exceeds a predetermined value, the set temperature of the microorganism sensor is set higher than the optimum growth temperature of the microorganism.

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