JPS6057537B2 - Ammonia determination method using microbial electrodes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying ammonia and ammonium ions using microbial chemistry. A microbial system that further oxidizes nitrite to nitric acid, or the oxygen partial pressure produced by the nitrite-forming action or the nitrite-forming action and the nitrifying action, which is caused by contacting the active bacterial cells of microorganisms that further oxidize nitrite to nitric acid, or both microorganisms. Ammonia and ammonium ions (hereinafter, both may be collectively referred to as ammonia) using microbial electrodes that electrochemically sense changes and thereby indirectly quantify the amount of ammonia in the test sample.

) and a device used therefor. While ammonia is an essential form of the nitrogen cycle in nature, it has recently been recognized as one of the causes of environmental pollution, causing eutrophication that destroys the environment of lakes and inner bays, and as a harmful substance to aquatic life. , there is a need to monitor ammonia in wastewater, and on the other hand, there is an extremely large need to quantify ammonia in process liquids as raw materials and by-products in the food industry, chemical industry, gas manufacturing industry, and textile industry. .

On the other hand, current methods for quantifying ammonia include distillation titration method (JISK1020), colorimetry method, and ammonia electrode method, but distillation titration method is performed manually and is not suitable for automation. For example, attempts have been made to automate methods such as measuring the absorbance after applying a salicylic acid coloring reagent or alkaline dichloroisocyanuric acid to purlins, but due to the need to completely remove solids from the purlins, It takes time and effort to change the paper, and the equipment is complicated (Annaka et al.: Continuous automatic analysis of phosphorus and ammonia nitrogen, "Water and Wastewater", 1, 48-54 (1978)) Automation method It is not necessarily appropriate.

In principle, the ammonia electrode method requires the pH of the water to be 11.0 or higher, which requires the injection of a large amount of alkali, and metal hydroxides deposit on the sensing surface of the detection unit, impairing measurement accuracy. There is a drawback that frequent cleaning of the membrane is required because of deterioration.

(Annaka et al.: Same as above) As a result of intensive research into a measurement method that is simple, stable, reliable, and suitable for online management of continuous methods, the present inventors have discovered that only ammonia can be selectively oxidized. A microbial electrode in which a layer of active microbial cells of nitric acid or a microbial system that oxidizes to nitric acid, or both, is immobilized with a thin film having micropores that do not allow the microbial cells to pass through, or active bacterial cells of the bacterium immobilized by a known method. We have discovered that a simple, stable, and reliable method for quantifying ammonia can be achieved by using a microbial electrode equipped with a microbial membrane immobilized with ammonia, and based on this discovery, we have completed the present invention. That is, the present invention provides a microbial electrode in which a layer of active bacterial cells of nitrite-producing bacteria or nitrite-producing bacteria and nitrate-producing bacteria is immobilized with a thin film having micropores that do not allow the bacterial cells to pass through, or A microbial electrode equipped with an immobilized microbial membrane is immersed in and brought into contact with a test solution containing ammonia and ammonium ions, and the amount of dissolved oxygen in the test solution is reduced in response to the ammonia concentration in the test solution. of,
A method for quantifying ammonia and ammonium ions using a microbial electrode, characterized in that the ammonia concentration is indirectly determined by sensing the amount of decrease in the output current of the microbial electrode or the rate of decrease in the output current, and an apparatus used therefor. This is the gist of the invention.

The microorganisms used in the present invention are called nitrifying bacteria, and are nitrite-producing bacteria that oxidize ammonia to nitrite, such as Nitrosomonas europaea (NitrO
sOmOnaseurOpaea) ATCCl97l&
Alternatively, nitrate-producing bacteria that further oxidize nitrite, such as NitrObacter
It is preferable to use nitrate-producing bacteria in combination with nitrite-producing bacteria because the inhibition of nitrite, which is an intermediate product, is less.

In addition to the above, examples of nitrite-producing bacteria include Nitrosomonas Monocera (NitrOsOmOnO
cella), Nitrosococcus●Nitrosus (Nit
rOsOcOccusnitrOsus), Nitrosospira briensis (NitrOsOspjrabri
Nitrosospira antarctica (N. ensis), Nitrosospira antarctica (N.
itrOsOspiraantartica), Nitrosocystei jabanensis (NitrOsOcy
stisjavarlensis), Nitrosocystis coccoides (NitrOsOcystiscOc)
cOides), Nitrosogloea Melismoides (
NitrOsOgIOeamerismOides),
Nitrosogloea Schizobacteroides (NitrOs)
OglOeashizObacterOides), Nitrosogloea Membranaceaea (NitrOsOg
Examples of nitrate-producing bacteria include Nitropacter and NitrObacterwinograds.
ki), Nitrocystis sarchinoides (Nitr
Ocystissarcir K) Ides), Nitrocystis microbanktate (NitrOcysti
smicrOpurlctate), etc.

These microorganisms utilize carbonate and bicarbonate as their sole carbon source and obtain the energy needed for growth by oxidizing ammonia or nitrite, so-called autotrophic bacteria.
), which oxidizes only ammonia or nitrous acid and consumes oxygen, and does not show any activity against coexisting organic compounds, so it is most suitable for the purpose of the present invention. That is, nitrite-producing bacteria generate energy through the following steps, and the reaction energy at this time causes the bacteria to proliferate.

Both are specific and cannot be performed interchangeably with the above reactions. Since both of these microorganisms exist in soil and activated sludge (particularly sludge that has been retained for a long period of time), they are usually separated from these for use.

As a medium for separation and culture, a medium such as Lenis's solution or Stephenson's solution may be used for culture and separation. According to this method, a mixture of both types of microorganisms is usually used.
Microbial electrodes manufactured using such nitrite-producing bacteria or nitrite-producing bacteria and nitrate-producing bacteria have a response to organic compounds such as glucose that is less than 1/1000 times the response to ammonia at the same concentration. The error caused by this can be ignored in practical terms.

Apart from the above methods of obtaining microbial sources from soil, sludge, etc.
Pure nitrifying bacteria, such as Nitrosomonas europaea ATCCl97l8, which is a bacterium of the genus Nitrosomonas, and/or Nitropacter agilis ATCCl4l23, which is a bacterium of the genus Nitropacter, was used as a seed and cultured in a medium containing nutrients such as ammonium sulfate and phosphoric acid. It can be carried out using

After culturing the nitrifying bacteria produced by any of these methods for several months using the method described above, calcium carbonate (magnesium carbonate or silicate gel may also be used in some cases) is added as a carrier to the culture solution.

These are called bacterial carriers). The active cells of the nitrifying bacteria that are attached and dispersed are separated and collected by a method such as filtration or centrifugation and used. The oxygen electrode used in the present invention may be either a polaro type or a galvanic type, and commonly available commercially available ones can be used.

An example of the microbial electrode used in the present invention is shown in FIG. 1st
The figure shows a front sectional view of a microbial electrode using a galvanic type oxygen electrode. The active bacterial cells used are the active bacterial cells isolated and collected as described above, or a paste of the bacterial cells adhered to calcium carbonate, which is made into a thin layer, supported by a nylon net if necessary, and then covered with micropores as described below. The microorganism electrode 11 shown in FIG. 1 can be obtained by fixing with a thin film having a rubber band or the like. In the figure, 1 is the microbial cell layer, 2
A support that covers and supports these, such as a nylon net,
3 is a thin film having micropores such as a porous membrane, dialysis membrane, or gas permeable membrane; 4 is a diaphragm of an oxygen electrode; 5 is a platinum cathode;
6 is an aluminum anode, 7 is a potassium chloride electrolyte, and 8 and 8'' are rubber bands for fixation.The thin film having micropores that covers and supports the microbial cell layer 1 is the thin film according to the present invention. Any thin film that does not pass through the cells of the microorganisms used but allows ammonia, ammonium ions, oxygen, etc. to freely pass through may be used, such as porous membranes such as millibore filters, cellophane, animal semipermeable membranes, etc. Any membrane that satisfies the above conditions can be used, such as a dialysis membrane, such as a gas permeable membrane such as a polyfluoroethylene resin membrane or a silicone membrane.

An immobilized microbial membrane may be used in place of the microbial layer 1 in Figure 1, and the immobilized microbial membrane is formed by immobilizing microorganisms with collagen or polyacrylamide gel, etc. in accordance with the usual enzyme entrapment immobilization method. It can also be used by attaching it to an electrode by an appropriate method, such as making it, cutting it to an appropriate size, and encapsulating it. Since there is almost no difference in activity or function whether an active microbial cell layer or an immobilized microbial membrane is used, there is no particular need to use an immobilized microbial membrane. In order to quantify ammonia using the microbial electrode of the present invention, when the microbial electrode 11 is immersed in and brought into contact with a test solution containing ammonia, ammonia and other components of the test solution and oxygen are transferred to a porous membrane having micropores. 3 and diffuses into the microbial layer 1.

Since microorganisms consume oxygen by oxidizing only ammonia, dissolved oxygen in the area near the oxygen electrode membrane 4 decreases in proportion to this, and as a result, the output current of the microorganism electrode decreases. . When the ammonia oxidation ability of microorganisms is sufficiently large compared to the amount of ammonia diffused through the porous membrane 3, that is, under diffusion-limited conditions, the decrease value or rate of decrease in the output current of the microbial electrode and the rate of decrease in the output current of the microbial electrode A proportional relationship is established between the ammonia concentration of the liquid and the ammonia concentration. Therefore, by measuring the amount or rate of decrease in the output current, the ammonia concentration in the test liquid can be determined. In order to obtain such a proportional relationship, it is desirable to apply a sufficient amount of microorganisms and to maintain the pH of the test solution at 7 to 9 in order to maintain its activity. Potassium may be added in an amount of about 0.05M01'e. To carry out the ammonia measurement of the present invention as a continuous operation, an apparatus such as that shown in the system assembly diagram of FIG. 2 may be used, for example. In the figure, 11 is the microbial electrode of the present invention, 12 is a rubber backing, 13 is a flow cell (inner volume 2 to 10 m1), 14 is a magnetic stirrer, 15 is a stirrer, 16 is a recorder, 17 is an air inlet, and 18 is a buffer Reference numeral 19 indicates a liquid (carrier liquid) injection port and a sample injection port. A case in which the method of the present invention is carried out continuously will be explained with reference to FIG.

First, a buffer solution (carrier solution) 18 saturated with oxygen is passed through the measurement cell (flow cell) 13 using a pump, and the test solution is injected one to one drop at a time using a sampler at regular intervals (for example, from 10 to 10 minutes). When injected from the inlet 19, the test liquid is appropriately diluted with the gearier liquid (ammonia concentration of several ppm), enters the flow cell 13, and enters the microbial electrode 1.
1, the ammonia in the test liquid is oxidized by the microbial layer, oxygen is consumed accordingly, the dissolved oxygen concentration near the oxygen electrode membrane decreases, and the output current of the oxygen electrode decreases. , are recorded on the recorder 16. If the relationship between the ammonia concentration in the test solution and the current reduction value (corresponding to the height of the peak on the recording paper) is determined for a known standard ammonia concentration solution, the ammonia concentration in the test solution can be determined from the peak height. can be easily found. When quantifying ammonia concentration in a batch method, the microbial electrode is immersed in and brought into contact with the test liquid, and the amount of decrease in output current over a certain period of time, for example within the range of 1 to 1 particle, is determined or measured. If the dissolved oxygen concentration of the test solution is kept constant by aerating the test solution, the output current value will become constant as the quantification time increases and an equilibrium state will be reached.The output current value at this equilibrium or When the rate of decrease of the output current is determined, since these values are proportional to the ammonia concentration in the test liquid, the concentration can be determined. In the case of quantitative determination, it is desirable to carry out the conditions in a highly active range depending on the type of microorganism used, and the temperature is usually 25 to 35°C and the pH is 7 to 7.
A range of 9 is preferred.

Since the reaction of microorganisms is fast, a contact time of 0.5 to 10 minutes between the microorganism electrode and the test liquid is sufficient. This short contact time between the test liquid and the microbial electrode not only shortens the quantification time, but also reduces the degree of contamination of the microbial electrode. Enable long-term use of the device. In addition, in addition to nitrifying bacteria, the microbial layer contains a small amount of microorganisms that assimilate and oxidize organic compounds, which causes a slight response to the organic compounds contained in the test liquid. , as mentioned earlier. By culturing nitrifying bacteria for several months, undesirable microorganisms other than nitrifying bacteria are eliminated.
For practical purposes, the response to coexisting organic compounds can be reduced to an acceptable extent.

(1/1000 or less compared to ammonia) Furthermore, in order to completely eliminate sensitivity to organic compounds, porous membrane 3
A membrane that selectively permeates only gases such as ammonia and oxygen, such as a porous Teflon (registered trademark) membrane or a silicone membrane, can be used as the membrane. In this case, ammonium ions in the test liquid can be gasified. In order to do this, the pH of the test solution is 9.
It is preferable to maintain the temperature at or above.

As stated above, the method for quantifying ammonia of the present invention is as follows:
Provides an industrially superior method and device for quantifying ammonia that enables selective, simple, and rapid measurement of ammonia concentration in factory wastewater and fermentation liquids in which other organic substances coexist. It is something to do.

Examples are shown below. Note that unless otherwise specified, % indicates weight %. Example 1 After filling 2.5 e of the medium A shown in Table 1 into an aeration tank with a volume of 5 e and adding 15 y of powdered calcium carbonate, the pH was adjusted to 8.0 with 1N soda carbonate, and activated sludge containing nitrifying bacteria was added to this. 200 ml (MLSS5OOOppm) was added, and air was blown in at 1/3 to 1/2 VM using an aeration pipe for aerobic cultivation.

When the nitrifying bacteria started to act, nitric acid was produced by oxidation of ammonia, and the pH decreased, so 1N soda carbonate was added to keep the pH at 8.0.

In this state, 200 ml ID of medium B was continuously supplied and cultured at room temperature (20 to 30°C). An overflow port was provided so that the culture solution in the aeration tank was always at 2.511 tre. After culturing for 4 months, 5 ml of the culture solution was filtered through a millibore filter with a diameter of 47 mm to allow microorganisms to adhere to the surface.

This membrane is pasted on the diaphragm of a galvanic-type oxygen electrode so that the microbial layer is sandwiched inside, the top is covered with a nylon net, and it is fixed to the oxygen electrode body with a rubber band to create a microbial electrode. The continuous measurement system shown in Figure 2 was assembled using electrodes and the following experiments were conducted. first,
0.05 M'e of soda and 0.05 M'e as carrier liquid.
A mixture of equal volumes of monopotassium phosphate (pH 8.05MIe)
8) is prepared, air is blown into it to saturate it with dissolved oxygen, and then it is continuously supplied to the flow cell at a flow rate of 3.9 mtImin.

Separately, air was sent in at a flow rate of 3.9 ml/min. At this time, the measurement cell was placed inside a heat insulation jacket (not shown in FIG. 2) and the temperature was maintained at 30°C. In this way, the output current of the microbial electrode was recorded on a recorder (baseline), and after the value became stable, the test liquid was sent to the flow cell at a flow rate of 0.8 ml/min, and the change in the output current of the microbial electrode was recorded. When the output current reached equilibrium, the supply of the test liquid was stopped and the output current was confirmed to recover to its initial value. Such a test was carried out using ammonium sulfate at 10, 20, 30
The test was conducted using a test solution in which Tn9 was dissolved in ion-exchanged water to prepare 111tre.

The results are shown in FIG. 3, where the vertical axis shows the output current in μA, and the horizontal axis shows time in minutes.

Curves 1, 2 and 3 in the figure are 10 and 20 of ammonium sulfate, respectively.
and 30m9 dissolved in ion-exchange treated water of 1e. 4 indicates the output current value of the ion exchange treated water.

In addition, Figure 4 was created by plotting the equilibrium current value of the microbial electrode against the ammonia concentration of the test liquid. The vertical axis shows the output current (μA), and the horizontal axis shows the ammonia concentration (Ppm). In addition, the test solutions of ammonium sulfate 10, 20, and 30 ppm solutions have ammonia concentrations of 2.58 and 5.15, respectively.
, 7.72 ppm. In the figure, 1, 2, and 3 are the plots of the output current of the ammonium sulfate solution in the above order, and 4 is the current plot of the ion-exchanged water. An equilibrium current is obtained,
Furthermore, as shown in FIG. 4, it was found that the equilibrium current value showed a good linear relationship with the ammonia concentration. Note that since the test liquid is diluted by about 5 times with the carrier liquid, the ammonia concentration in the measurement cell is between 0.5 and 1.
5 ppm, indicating that this microbial electrode has extremely high sensitivity. Next, in order to investigate the influence of organic substances, we measured a sample solution containing 1000 ppm of glucose, and the response was 10 ppm of ammonium sulfate (2.58 ppm in terms of ammonia).
ppm) of the test solution. That is, this microbial electrode showed a sensitivity of about 2×10 3 times as much for ammonia as for glucose, and showed sufficient selectivity for practical use. When this measurement system was operated continuously for more than 10 days, it operated stably and the response characteristics to ammonia remained unchanged.

However, the sensitivity ratio of ammonia to the above-mentioned glucose-containing test solution was 1×1σ times, and although the selectivity for ammonia was slightly lowered, it was still sufficient for practical use. Example 2 A microbial electrode and a quantitative device prepared in the same manner as in Example 1 were operated under the same conditions to actually measure ammonia in waste water.

As wastewater, four types of fermentation factory wastewater as shown in Table 2 were diluted with ion-exchanged water and quantified.

The free ammonia concentration in the table is JISKOlO2 (Japanese Industrial Standards Committee: Factory wastewater test method K.OlO2, P3
6), the BOD is also JISK. O
Each was measured by the method specified in 1O2 (Test method KO1O2, P33).

Separately, as a standard substance, ammonium sulfate solution was used as in Example 1, and the ammonia concentration was 2.58, 5.15, 7.72.
A ppm standard solution was prepared.

The above test liquid was injected into the measuring cell at a flow rate of 0.877!11 Min, one drop at a time every three drops.

The results are shown in FIG. 5 (output current (μA) on the vertical axis and ammonia concentration (Ppm) on the horizontal axis). White circles 1, 2, and 3 in the figure are plots of the relationship between the ammonia concentration of the standard solution and the output current value of the electrode, and there exists a linear relationship between the two as shown in the figure. Next, the ammonia concentration of the test liquid was similarly determined using the distillation titration method, and the same liquid was measured using a microbial electrode. When the relationship between the measured output currents was plotted, the results were as shown by black circles 5 to 8 in FIG. These black circles 5
-8 are all located near the standard solution straight line. This shows that the concentration measured with the microbial electrode and the concentration determined by distillation titration are in good agreement. Example 3 The culture medium shown in Table 3 was transferred to a 2000 ml Erlenmeyer flask, and after adjusting the pH to 8.2 to 8.4 with a 50% potassium carbonate solution, it was sterilized at 120° C. for three times.

However, in order to prevent the formation of precipitates, magnesium sulfate and calcium chloride were separately sterilized under the same conditions as above, and then mixed after being allowed to cool.

Nitrosomonas europaea (Ni
tr′0s0m0naseur0paea)Aπ℃19
718 culture solution was inoculated and incubated at room temperature (around 30℃) for 2 hours.
It was statically cultured for 5 days.

If the pH of the indicator cresol red changes from red to yellow during culturing, drop a sterilized 50% potassium carbonate solution until the indicator returns to its original color.
was kept constant.

The total volume of the culture solution thus obtained was 47TfI in diameter! A microbial electrode was assembled in the same manner as in Example 1 using a membrane on which microorganisms were attached to the surface of the membrane filtered through a millibore filter, and operated under the same conditions and method.

As a result, as shown in Table 4, the output current decreased in proportion to the ammonia concentration. In addition, ammonium sulfate 100,
The 200 and 400 ppm solutions are converted into ammonia concentrations of 25.8, 51.5 and 77.2 ppm, respectively.

[Brief explanation of drawings]

FIG. 1 is a front longitudinal sectional view of the structure of the microbial electrode of the present invention. In the figure: 1 bacterial cell layer, 2 support, 3 porous membrane, 4 oxygen electrode diaphragm, 5 cathode, 6 anode, 7 potassium chloride electrolyte,
8 and 8" are ring combs, and Figure 2 is an explanatory diagram of the assembly of a continuous quantitative device system using the microbial electrode of the present invention. In the figure, 11 microbial electrode, 12 rubber backing, 13 flow cell, 14 magnetic stirrer, and 15 stirrer. ,1
6 recorders, 17 air inlets, 18 carrier inlets, 19 sample inlets.

Claims (1)

[Claims] 1. A microbial electrode in which a layer of active bacterial cells of nitrite-producing bacteria or nitrite-producing bacteria and nitrate-producing bacteria is immobilized on an oxygen electrode with a thin film having micropores that do not allow the bacteria to pass through, or A microbial electrode equipped with an immobilized microbial membrane is immersed in and brought into contact with a test solution containing ammonia and ammonium ions, and a decrease in dissolved oxygen in the test solution that occurs in response to the ammonia concentration of the test solution is detected. A method for quantifying ammonia and ammonium ions using a microbial electrode, characterized in that the ammonia concentration is indirectly determined by sensing the amount of decrease in the output current or the rate of decrease in the output current of the microbial electrode.