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Development of long-term stable ammonium ion sensor in conjunction with a microbial membrane
Biosensors & Bioelectronics 13 (1998) 531–537
Development of long-term stable ammonium ion sensor in conjunction with a microbial membrane Manami Ikeda a, Hiromitsu Hachiya a, Satoshi Ito a, Yasukazu Asano Toshihiko Imato c
b,*
,
a b
DKK Corporation, Kitamachi Kichijoji Musashino, Tokyo 180-0001, Japan Ariake National College of Technology, Higashiogio, Omuta 836-8585, Japan c Kyushu University, Hakozaki Higashi-ku, Fukuoka 812-0053, Japan Received in revised form 14 March 1997; accepted 20 October 1997
Abstract An ammonium ion sensor, stable over a long time frame, was developed for the continuous monitoring of ammonium ion in sewage. The sensor consists of a plasticized poly (vinyl chloride) membrane-based ammonium ion sensor, in which the sensing membrane was covered with a microbial membrane (microorganism: Trichosporon cutaneum). The developed ammonium ion sensor completely eliminated interference from cationic surfactants as the result of attaching the microbial membrane to the sensing membrane. The sensor could be utilized for continuous monitoring in sewage for over 120 days, while the performance of the ammonium ion sensor without the microbial membrane deteriorated within 5 days. The improvement of long-term stability of the developed sensor is due to the fact that Trichosporon cutaneum in the microbial membrane effectively assimilated organic compounds, such as ionic surfactants, from the sewage and the sensing membrane of the sensor was prevented from coming into direct contact with organic compounds. A regression line of Y = 1.01X + 0.962 × 10⫺4 with a correlation factor of 0.999 was obtained between the analytical results of the present sensor method (Y) and those of a conventional method (X) for the determination of ammonium ion in a sewage sample. 1998 Elsevier Science S.A. All rights reserved. Keywords: Potentiometry; PVC membrane; Ammonium ion sensor; Nonactin; Microbial membrane; Sewage
1. INTRODUCTION Two types of potentiometric ammonium ion sensors are presently commercially available. One is an ammonium ion sensor based on a gas sensor which consists of a pH glass membrane sensor and a gas-permeable membrane. Using this sensor, it is necessary to add an alkaline solution to the sample solution, in order to evolve ammonia. The evolved ammonia is then detected by the pH glass membrane sensor, after permeation through the gas-permeable membrane. The second sensor consists of a plasticized poly (vinyl chloride) (PVC) membrane sensor, the membrane of which contains nonactin as an ionophore. This sensor is capable of the direct determination of ammonium ion without any prior pretreatment. This latter sensor has been
* Author to whom correspondence should be addressed. 0956-5663/98/$19.00 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 9 7 ) 0 0 1 2 0 - 6
widely used for monitoring ammonium ion in the waste water, since it has a number of advantages over the gas sensor-based ammonium sensor, including cost efficiency, ease of handling, and no need for adjustment of the pH of sample solutions (Ammann, 1985; Ikeda et al., 1987; Ikeda et al., 1988). However, it is well known that the potential response of this type of sensor, based on a PVC membrane, is subject to interference by anionic surfactants (Frend et al., 1983) as well as hydrophobic substances (Shibata et al., 1992; Shibata et al., 1993). Indeed, our preliminary experiments show that the life-time of the PVC membrane based ammonium ion sensor in sewage is very short. This may be due to the fact that the sensing membrane deteriorates on contact with sewage water which contains organic compounds, such as ionic surfactants and alcohols, and as a result, the sensing function of the membrane may be lost. This phenomenon has been often observed for other PVC membrane-based sensors. It has been concluded
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that the deterioration of the PVC membrane-based sensors is caused by the solubilization of a plasticizer and an ionophore from the sensing membrane into the sample solution, when it contains lipid-soluble organic substances, such as organic anions (Hulanicki et al., 1982), ionic surfactants (Masadome et al., 1992) and alcohols (Anzai and Liu, 1991). Biological oxygen demand (BOD) is a critical indicator in the evaluation of pollution in environmental water. The conventional 5 day BOD method to determine the BOD of waste waters has been displaced with a microbial sensor method because of its ability to rapidly determine BOD (Japanese Industrial Standard, 1991; Marty et al., 1997). The principle of the BOD sensor is based on the determination of the amount of oxygen consumed by a microorganism (Trichosporon cutaneum), which is immobilized in the membrane, and which is proportional to BOD when the immobilized microorganism assimilates the organic compounds in the sample solution (Karube et al., 1977; Hikuma et al., 1979). It is expected that the life-time of the PVC membranebased ammonium ion sensor could be improved providing the sensor membrane is covered with a microbial membrane, similar to the BOD sensor since such a sensor membrane would be prevented from coming into contact with organic compounds in the sample solution directly. Based on this rational, we report the development of a long-term stable ammonium ion sensor which contains a microbial membrane which eliminates interference from organic substances, such as dodecyltrimethylammonium ion. In this paper, we report on the performance of such a sensor and its application to sewerage.
0.3% malt extract, 0.3% polypeptone, 0.3% yeast extract and 1% glucose under aerobic conditions at 30°C for 36 h. The original broth was diluted with 26 volumes of deionized water whose optical density, measured at a wavelength of 562 nm was 0.33. 3 ml of the diluted culture broth was dropped into a 6 mm I. D. hole of a 1 mm thick plastic spacer which had been placed on an cellulose acetate membrane (pore size: 0.45 m, thickness: 0.1 mm) under suction with an aspirator. The microorganism was immobilized onto the cellulose acetate membrane in the hole. The top of the hole was covered with the another cellulose acetate membrane and the spacer, along with the two cellulose acetate membranes were fasten together with an adhesive. This assembly comprised the microorganism immobilized membrane. 2.3. Preparation of PVC membrane for ammonium ion sensor A plasticized poly (vinyl chloride) (PVC) membrane was prepared as follows: Plasticizer (dioctylsebacate, 1.0 g), PVC (2.0 g) and nonactin (15 mg) were dissolved in 8 ml of tetrahydrofuran (THF). 10 l of the THF solution was added dropwise onto a porous PTFE membrane which was placed on an end of a sensor chip (DKK, Sensor Kit) and the THF was then allowed to evaporate. This procedure was repeated a total of 30 times. Finally, the sensor chip was maintained at 30°C for 12 h, giving the plasticized PVC membrane on the sensor chip. The concentration of nonactin in the plasticized PVC membrane thus obtained is approximately 2.0 × 10⫺2 mol l⫺1 of plasticizer. The thickness of the membrane is ca. 0.5 mm. 2.4. Assembly of ammonium ion sensor with microbial membrane
2. EXPERIMENTAL 2.1. Reagents All chemicals were of analytical reagent grade. Nonactin and the microorganism (yeast; Trichosporon cutaneum AJ 4816) were obtained from Fluka Co. Ltd. and the National Institute of Bioscience and Human-Technology (Patent Microorganism Depository Division), respectively. Pure water which had been treated with a 0.45 m membrane filter (electric conductivity ⬍ 0.1 S cm⫺1 was used throughout the work.
The structure of the ammonium ion sensor, along with the microbial membrane is shown in Fig. 1. The sensor chip with the PVC membrane was filled with 3 ml of 0.01 mol l⫺1 ammonium chloride solution as the inner filling solution. The sensor chip was then screwed into a sensor body consisting of an inner chloride ion electrode and a reference electrode with the porous PTFE liquid junction (Ito et al., 1996). The microbial membrane was fixed on the surface of the PVC membrane so that the two were in close contact.
2.2. Culture and immobilization of microorganisms
2.5. Response mechanism of ammonium ion sensor with microbial membrane
Culture and immobilization of the microorganism were done essentially according to Hikuma’s procedure (Hikuma et al., 1979). 10 mg of the microorganism, which had been stored in a refrigerator at 10°C was cultured in 50 ml of a culture medium (pH 6.0) containing
When the ammonium ion sensor with the microbial membrane is immersed in a sample solution containing ammonium ion and organic compounds, organic compounds, which diffuse into the microbial membrane are assimilated by the microorganism in the membrane and
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Fig. 1. Structure of the PVC membrane-based ammonium ion sensor with microbial membrane. 1. Terminal, 2. Sensor cap, 3. PTFE body, 4. Reference electrode, 5. Sensor chip, 6. Plasticized PVC membrane, 7. Microbial membrane, 8. Supporter for microbial membrane, 9. Ammonium chloride inner solution, 10. Inner chloride ion electrode, 11. Saturated potassium chloride paste.
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are then decomposed. However, the ammonium ion in the sample solution permeates through the microbial membrane without assimilation and reaches the sensing membrane of the PVC membrane-based ammonium ion sensor. As a result, ammonium ion in the sample solution can be determined by the ammonium ion sensor, in conjunction with the microbial membrane without interference from organic compounds. 2.6. Apparatus The potential of the ammonium ion sensor with the microbial membrane was measured using an ion meter (DKK: IOL-50). The experimental data were recorded on a chart recorder (SEKONIC: 250F) and stored in a calculator (HP: 41C). A PVC membrane-based ammonium ion sensor (DKK: 7494L) and a reference electrode (DKK: 4083) were used for comparison with the sensor developed in this work.
Fig. 2. Calibration curve for ammonium ion. 쐌 PVC membrane-based ammonium ion sensor with microbial membrane. 왕 PVC membranebased ammonium ion sensor without microbial membrane. Samples: Standard solution (pH 6.0, 25°C).
3. RESULTS AND DISCUSSION 3.1. Calibration curve The relationship between the concentration of ammonium ion and the potential of the PVC membranebased ammonium ion sensor in conjunction with the microbial membrane was investigated for a series of standard NH4Cl solutions of different concentrations. The pH of the standard solutions was adjusted to pH 6.0 using a lithium acetate buffer solution. The data were compared with those obtained with the PVC membranebased ammonium ion sensor in the absence of the microbial membrane. The same calibration curves with a near Nernst slope of 56 mV/decade were obtained for both sensors with and without the microbial membrane over the concentration range from 10⫺4 to 10⫺1 mol l⫺1 of ammonium ion, as shown in Fig. 2. This indicates that the microbial membrane had no effect on the potential of the PVC membrane-based ammonium ion sensor, for the case where the sample solution contained no organic compounds. The lower detection limit of the sensor with the microbial membrane was 5 × 10⫺5 mol 1⫺1, and was slightly higher than the sensor without the microbial membrane. The reproducibility of potentials of the sensor with the microbial membrane was 8.3% as a standard deviation for 5 times measurements of the 10⫺4 mol l⫺1 standard solution. 3.2. Response time Figure 3 shows the transient response of the PVC membrane-based ammonium ion sensor with the microbial membrane, when the concentration of ammonium ion was changed from 10⫺4 to 10⫺3 mol l⫺1.
Fig. 3. Response time of PVC membrane-based ammonium ion sensor in conjunction with microbial membrane. Sample: 10⫺4 → 10⫺3 mol l⫺l ammonium chloride solution.
In this case, the pH of the sample solution was also adjusted to pH 6.0 with a lithium acetate buffer solution. The potential of the sensor increases gradually with time, and finally reaches a constant potential. The response time, defined as the time require for the potential to reach 90% of the final value, was 6 min. Since the response time of the PVC membrane-based ammonium ion sensor without the microbial membrane was 10 sec, the response time of the developed ammonium sensor with the microbial membrane is fairly slow, This slow response may be due to the slow diffusion of ammonium ion from the sample solution to the surface of the PVC membrane across the microbial membrane.
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3.3. Effectiveness of microbial membrane against organic compounds As described in the section on response mechanisms, if the microbial membrane functions effectively, the present sensor will not be subject to interference from organic compounds. To examine effectiveness of the microbial membrane against organic compounds, the potential response of the ammonium ion sensor with and without the microbial membrane was examined for three standard solutions of ammonium ion (10⫺4, 10⫺3 and 10⫺2 mol l⫺1) and the same standard solutions containing 10⫺5 mol l⫺1 dodecytrimethylammonium ion (DOTA). DOTA was chosen as a typical organic compound found in waste water. The potentials of the sensor with and without the microbial membrane were measured for the above solutions and the results are shown in Fig. 4. A good linear relationship between the potential and the logarithmic concentration of ammonium ion was obtained for both sensors. However, the potential response of the sensor without the microbial membrane was shifted by 250 mV in a positive direction for the standard solution containing DOTA, compared to the standard solution without DOTA. In addition, the potential response of the sensor with the microbial membrane to the standard solution containing DOTA was nearly the same as for the standard solution without DOTA. This strongly suggests that DOTA was assimilated by the immobilized Trichosporon cutaneum in the microbial membrane when DOTA and ammonium ion diffused into the microbial membrane and that the interference from DOTA was eliminated. The assimilation rate of Trichosporon cutaneum for DOTA at the present
Fig. 4. Effectiveness of the microbial membrane against organic compound. Sample: standard ammonium ion solution. ⴰ The conventional sensor. • The present sensor with a microbial membrane. Sample: standard ammonium solution containing 10⫺5 mol l⫺1 dodecyltrimethyl ammonium ion. 왕 The conventional sensor. 왖 The present sensor with a microbial membrane.
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concentration would be predicted to be sufficiently faster than the diffusion rate of DOTA through the microbial membrane. The assimilation ability of the microbial membrane may be effective for other organic compounds judging from data on the BOD sensor fabricated with the same microbial membrane (Karube et al., 1977; Hikuma et al., 1979; Marty et al., 1997). 3.4. Correlation between the present sensor method and the Japanese Industrial Standard method for determination of ammonium ion in sewage samples The present sensor was applied to the determination of ammonium ion in sewage. The analytical results obtained by this method were correlated with data obtained by the Japanese Industrial Standard method (JIS: Indophenol method) (Japanese Industrial Standard, 1991). The correlation data are shown in Fig. 5. A regression line expressed by Y = 1.01X + 0.963x10⫺4 with a correlation factor of 0.999 between both methods was obtained if the result for the sample which contained ammonium ion at 10⫺2 mol l⫺1 is omitted. Fig. 5 indicates that the analytical results by the present sensor method are in good agreement with those by the JIS method for samples containing ammonium ion concentration of less than 5 × 10⫺3 mol l⫺1. However, the analytical result for the sample which contained about 1 × 10⫺2 mol l⫺1 as obtained by the present sensor method was higher than that by the JIS method by 20%. This discrepancy may arise from the fact that the activity of the microorganism in the microbial membrane is deactivated by these high levels of ammonium ion and, thus, organic compounds in waste water may interfere with the sensor. Such deactivation of microorganisms by inorganic ions is often observed in similar biosensors (Karube et al., 1977). The present sensor method is
Fig. 5. Correlation between the present method and Japanese Industrial Standard Method. Samples: Sewage waste water. Regression line: Y = 1.01X + 0.962 × 10⫺4. Correlation factor: r = 0.999.
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clearly practical for the determination of ammonium ion at the concentration less than 5 × 10⫺3 mol l⫺1 in sewage. 3.5. Long-term stability in sewage The long-term stability of the ammonium ion sensor with the microbial membrane, together with the conventional ammonium ion sensor without the microbial membrane, was investigated during analysis of sewage over a period of 130 days. Both sensors were immersed in the sewage sample during this period. The potential response of the sensor was measured for two standard solutions of 10⫺4 and 10⫺2 mol l⫺1 ammonium chloride at regular intervals, and the potential slope against the logarithmic concentration of ammonium ion was then obtained. As shown in Fig. 6, the potential slope of the ammonium ion sensor without the microbial membrane rapidly decreased after 5 days of use. However, the ammonium ion sensor with the microbial membrane maintained its initial performance with a near Nernst slope (56 mV/decade) over the entire 120 day period. This suggests that there was no deterioration in performance of this sensor during this period, even for the case of the sewage sample. We attribute this to the fact that the microbial membrane completely prevents organic compounds from coming into contact with the sensing PVC membrane. This experiments also shows that the use of the membrane, immobilized with the aerobic microorganism (Trichosporon cutaneum), significantly increases the life-time of the PVC membrane-based ammonium sensor.
Fig. 6. Life-time test of ammonium ion sensors with and without microbial membrane in sewage sample. -쐌- with microbial membrane ⴰ- without microbial membrane.
4. CONCLUSIONS We have developed a new type of PVC membranebased ammonium ion sensor with a microbial membrane, in which Trichosporon cutaneum is immobilized. This sensor proved to be durable over a long period of continuous use in sewage analysis. The effective elimination of interference from organic compounds was found to be due to the ability of the microorganism to assimilate organic compounds. As a result, compared with the conventional ammonium ion sensor without the microbial membrane, the lifetime of the present sensor in sewage was increased from 5 to 120 days. Furthermore, the determination of ammonium ion in the presence of 10⫺5 mol l⫺1 dodecytrimethylammonium ion could be carried out using the present sensor method. The present sensor was applicable to the determination of ammonium ion in sewage. A good correlation (r = 0.999) was obtained between the analytical results obtained by the present sensor method and those by the conventional method. We conclude that the present sensor method has advantages of simplicity, low cost and ease of maintenance for the direct continuous monitoring for ammonium ion. The basic concept, namely the elimination of interference from organic compounds by use of the microbial membrane may be applicable to other potentiometric PVC membrane-based sensors.
References Ammann D. (1985). Ion-selective micro-electrodes, 2-32, SpringerVerlag. Anzai, J., Liu, C., 1991. Potentiometric response of ply(vinyl chloride)valinomycine membrane potassium ion sensors to no-ionic alcohols. Anal. Chim. Acta 248–, 323–327. Frend, A.J., Moody, G.J., Thomas, J.D.R., Birch, B.J., 1983. Study of calcium ion-selective electrode in the presense of anionic surfactants. Analyst 108–, 1072–1078. Hikuma, M., Suzuki, H., Yasuda, T., Karube, I., Suzuki, S., 1979. Amperometric estimation of BOD by using living immobilized yeasts. Euro. J. Appl. Microbiol. Biotechnol. 8–, 289–297. Hulanicki, H., Trojanowicz, M., Pobozy, E., 1982. Effect of surfactants on the response of ion-selective electrodes with poly(vinyl chloride) membranes. Analyst 107–, 1356–1362. Ikeda M., Ito S., Eto M. and Asano Y. (1987). Improvement of ammonium ion selective electrode using microbial membrane, The 48th the Annual Discussion Meeting of Japan Association for Analytical Chemistry, Abstract, 397-398. Ikeda M., Ito S. and Asano Y. (1988). Electrodes. Kokai Tokkyo Koho, 302355, Japan Patent Agency. Ito, S., Kobayashi, F., Baba, K., Asano, Y., Wada, H., 1996. Development of long-term stable reference electrode with fluoric resin liquid junction. Talanta 43–, 135–142. Japanese Industrial Standard JIS K-3602.(1991). Instrument for BOD with microbial electrode, Japanese Standards Association. Karube, K., Mitsuda, S., Matsunaga, T., Suzuki, S., 1977. A rapid method for estimation of BOD by using immobilized microbial cells. J. Ferm. Tech. 55–, 243–248. Marty, J.L., Olive, D., Asano, Y., 1997. Measurement of BOD: Corre-
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lation between 5 day BOD and commercial BOD biosensor values. Environ. Tech. 18–, 333–337. Masadome, T., Yang, J., Imato, T., Ishibashi, N., 1992. Anion response characteristics of an electrode based on plasticized poly(vinyl chloride) membrane without added ion exchanger. Bunseki Kagaku 41–, 231–237. Shibata Y., Maruizumi T., and Miyagi H. (1992). Characteristics of
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sodium ion-selective electrodes based on neutral carriers and their application to analysis of physiological fluids. Nippon Kagakukaishi, 961-967. Shibata, Y., Hirota, K., Miyagi, H., Takada, Y., 1993. Interfering substances in the analysis of patient serum samples with dialysis by chloride ion-selective polymeric membrane electrodes based on methyltridodecylammonium salt. Bunseki Kagaku 42–, 265–271.
Development of long-term stable ammonium ion sensor in conjunction with a microbial membrane Manami Ikeda a, Hiromitsu Hachiya a, Satoshi Ito a, Yasukazu Asano Toshihiko Imato c
b,*
,
a b
DKK Corporation, Kitamachi Kichijoji Musashino, Tokyo 180-0001, Japan Ariake National College of Technology, Higashiogio, Omuta 836-8585, Japan c Kyushu University, Hakozaki Higashi-ku, Fukuoka 812-0053, Japan Received in revised form 14 March 1997; accepted 20 October 1997
Abstract An ammonium ion sensor, stable over a long time frame, was developed for the continuous monitoring of ammonium ion in sewage. The sensor consists of a plasticized poly (vinyl chloride) membrane-based ammonium ion sensor, in which the sensing membrane was covered with a microbial membrane (microorganism: Trichosporon cutaneum). The developed ammonium ion sensor completely eliminated interference from cationic surfactants as the result of attaching the microbial membrane to the sensing membrane. The sensor could be utilized for continuous monitoring in sewage for over 120 days, while the performance of the ammonium ion sensor without the microbial membrane deteriorated within 5 days. The improvement of long-term stability of the developed sensor is due to the fact that Trichosporon cutaneum in the microbial membrane effectively assimilated organic compounds, such as ionic surfactants, from the sewage and the sensing membrane of the sensor was prevented from coming into direct contact with organic compounds. A regression line of Y = 1.01X + 0.962 × 10⫺4 with a correlation factor of 0.999 was obtained between the analytical results of the present sensor method (Y) and those of a conventional method (X) for the determination of ammonium ion in a sewage sample. 1998 Elsevier Science S.A. All rights reserved. Keywords: Potentiometry; PVC membrane; Ammonium ion sensor; Nonactin; Microbial membrane; Sewage
1. INTRODUCTION Two types of potentiometric ammonium ion sensors are presently commercially available. One is an ammonium ion sensor based on a gas sensor which consists of a pH glass membrane sensor and a gas-permeable membrane. Using this sensor, it is necessary to add an alkaline solution to the sample solution, in order to evolve ammonia. The evolved ammonia is then detected by the pH glass membrane sensor, after permeation through the gas-permeable membrane. The second sensor consists of a plasticized poly (vinyl chloride) (PVC) membrane sensor, the membrane of which contains nonactin as an ionophore. This sensor is capable of the direct determination of ammonium ion without any prior pretreatment. This latter sensor has been
* Author to whom correspondence should be addressed. 0956-5663/98/$19.00 1998 Elsevier Science S.A. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 9 7 ) 0 0 1 2 0 - 6
widely used for monitoring ammonium ion in the waste water, since it has a number of advantages over the gas sensor-based ammonium sensor, including cost efficiency, ease of handling, and no need for adjustment of the pH of sample solutions (Ammann, 1985; Ikeda et al., 1987; Ikeda et al., 1988). However, it is well known that the potential response of this type of sensor, based on a PVC membrane, is subject to interference by anionic surfactants (Frend et al., 1983) as well as hydrophobic substances (Shibata et al., 1992; Shibata et al., 1993). Indeed, our preliminary experiments show that the life-time of the PVC membrane based ammonium ion sensor in sewage is very short. This may be due to the fact that the sensing membrane deteriorates on contact with sewage water which contains organic compounds, such as ionic surfactants and alcohols, and as a result, the sensing function of the membrane may be lost. This phenomenon has been often observed for other PVC membrane-based sensors. It has been concluded
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that the deterioration of the PVC membrane-based sensors is caused by the solubilization of a plasticizer and an ionophore from the sensing membrane into the sample solution, when it contains lipid-soluble organic substances, such as organic anions (Hulanicki et al., 1982), ionic surfactants (Masadome et al., 1992) and alcohols (Anzai and Liu, 1991). Biological oxygen demand (BOD) is a critical indicator in the evaluation of pollution in environmental water. The conventional 5 day BOD method to determine the BOD of waste waters has been displaced with a microbial sensor method because of its ability to rapidly determine BOD (Japanese Industrial Standard, 1991; Marty et al., 1997). The principle of the BOD sensor is based on the determination of the amount of oxygen consumed by a microorganism (Trichosporon cutaneum), which is immobilized in the membrane, and which is proportional to BOD when the immobilized microorganism assimilates the organic compounds in the sample solution (Karube et al., 1977; Hikuma et al., 1979). It is expected that the life-time of the PVC membranebased ammonium ion sensor could be improved providing the sensor membrane is covered with a microbial membrane, similar to the BOD sensor since such a sensor membrane would be prevented from coming into contact with organic compounds in the sample solution directly. Based on this rational, we report the development of a long-term stable ammonium ion sensor which contains a microbial membrane which eliminates interference from organic substances, such as dodecyltrimethylammonium ion. In this paper, we report on the performance of such a sensor and its application to sewerage.
0.3% malt extract, 0.3% polypeptone, 0.3% yeast extract and 1% glucose under aerobic conditions at 30°C for 36 h. The original broth was diluted with 26 volumes of deionized water whose optical density, measured at a wavelength of 562 nm was 0.33. 3 ml of the diluted culture broth was dropped into a 6 mm I. D. hole of a 1 mm thick plastic spacer which had been placed on an cellulose acetate membrane (pore size: 0.45 m, thickness: 0.1 mm) under suction with an aspirator. The microorganism was immobilized onto the cellulose acetate membrane in the hole. The top of the hole was covered with the another cellulose acetate membrane and the spacer, along with the two cellulose acetate membranes were fasten together with an adhesive. This assembly comprised the microorganism immobilized membrane. 2.3. Preparation of PVC membrane for ammonium ion sensor A plasticized poly (vinyl chloride) (PVC) membrane was prepared as follows: Plasticizer (dioctylsebacate, 1.0 g), PVC (2.0 g) and nonactin (15 mg) were dissolved in 8 ml of tetrahydrofuran (THF). 10 l of the THF solution was added dropwise onto a porous PTFE membrane which was placed on an end of a sensor chip (DKK, Sensor Kit) and the THF was then allowed to evaporate. This procedure was repeated a total of 30 times. Finally, the sensor chip was maintained at 30°C for 12 h, giving the plasticized PVC membrane on the sensor chip. The concentration of nonactin in the plasticized PVC membrane thus obtained is approximately 2.0 × 10⫺2 mol l⫺1 of plasticizer. The thickness of the membrane is ca. 0.5 mm. 2.4. Assembly of ammonium ion sensor with microbial membrane
2. EXPERIMENTAL 2.1. Reagents All chemicals were of analytical reagent grade. Nonactin and the microorganism (yeast; Trichosporon cutaneum AJ 4816) were obtained from Fluka Co. Ltd. and the National Institute of Bioscience and Human-Technology (Patent Microorganism Depository Division), respectively. Pure water which had been treated with a 0.45 m membrane filter (electric conductivity ⬍ 0.1 S cm⫺1 was used throughout the work.
The structure of the ammonium ion sensor, along with the microbial membrane is shown in Fig. 1. The sensor chip with the PVC membrane was filled with 3 ml of 0.01 mol l⫺1 ammonium chloride solution as the inner filling solution. The sensor chip was then screwed into a sensor body consisting of an inner chloride ion electrode and a reference electrode with the porous PTFE liquid junction (Ito et al., 1996). The microbial membrane was fixed on the surface of the PVC membrane so that the two were in close contact.
2.2. Culture and immobilization of microorganisms
2.5. Response mechanism of ammonium ion sensor with microbial membrane
Culture and immobilization of the microorganism were done essentially according to Hikuma’s procedure (Hikuma et al., 1979). 10 mg of the microorganism, which had been stored in a refrigerator at 10°C was cultured in 50 ml of a culture medium (pH 6.0) containing
When the ammonium ion sensor with the microbial membrane is immersed in a sample solution containing ammonium ion and organic compounds, organic compounds, which diffuse into the microbial membrane are assimilated by the microorganism in the membrane and
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Fig. 1. Structure of the PVC membrane-based ammonium ion sensor with microbial membrane. 1. Terminal, 2. Sensor cap, 3. PTFE body, 4. Reference electrode, 5. Sensor chip, 6. Plasticized PVC membrane, 7. Microbial membrane, 8. Supporter for microbial membrane, 9. Ammonium chloride inner solution, 10. Inner chloride ion electrode, 11. Saturated potassium chloride paste.
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are then decomposed. However, the ammonium ion in the sample solution permeates through the microbial membrane without assimilation and reaches the sensing membrane of the PVC membrane-based ammonium ion sensor. As a result, ammonium ion in the sample solution can be determined by the ammonium ion sensor, in conjunction with the microbial membrane without interference from organic compounds. 2.6. Apparatus The potential of the ammonium ion sensor with the microbial membrane was measured using an ion meter (DKK: IOL-50). The experimental data were recorded on a chart recorder (SEKONIC: 250F) and stored in a calculator (HP: 41C). A PVC membrane-based ammonium ion sensor (DKK: 7494L) and a reference electrode (DKK: 4083) were used for comparison with the sensor developed in this work.
Fig. 2. Calibration curve for ammonium ion. 쐌 PVC membrane-based ammonium ion sensor with microbial membrane. 왕 PVC membranebased ammonium ion sensor without microbial membrane. Samples: Standard solution (pH 6.0, 25°C).
3. RESULTS AND DISCUSSION 3.1. Calibration curve The relationship between the concentration of ammonium ion and the potential of the PVC membranebased ammonium ion sensor in conjunction with the microbial membrane was investigated for a series of standard NH4Cl solutions of different concentrations. The pH of the standard solutions was adjusted to pH 6.0 using a lithium acetate buffer solution. The data were compared with those obtained with the PVC membranebased ammonium ion sensor in the absence of the microbial membrane. The same calibration curves with a near Nernst slope of 56 mV/decade were obtained for both sensors with and without the microbial membrane over the concentration range from 10⫺4 to 10⫺1 mol l⫺1 of ammonium ion, as shown in Fig. 2. This indicates that the microbial membrane had no effect on the potential of the PVC membrane-based ammonium ion sensor, for the case where the sample solution contained no organic compounds. The lower detection limit of the sensor with the microbial membrane was 5 × 10⫺5 mol 1⫺1, and was slightly higher than the sensor without the microbial membrane. The reproducibility of potentials of the sensor with the microbial membrane was 8.3% as a standard deviation for 5 times measurements of the 10⫺4 mol l⫺1 standard solution. 3.2. Response time Figure 3 shows the transient response of the PVC membrane-based ammonium ion sensor with the microbial membrane, when the concentration of ammonium ion was changed from 10⫺4 to 10⫺3 mol l⫺1.
Fig. 3. Response time of PVC membrane-based ammonium ion sensor in conjunction with microbial membrane. Sample: 10⫺4 → 10⫺3 mol l⫺l ammonium chloride solution.
In this case, the pH of the sample solution was also adjusted to pH 6.0 with a lithium acetate buffer solution. The potential of the sensor increases gradually with time, and finally reaches a constant potential. The response time, defined as the time require for the potential to reach 90% of the final value, was 6 min. Since the response time of the PVC membrane-based ammonium ion sensor without the microbial membrane was 10 sec, the response time of the developed ammonium sensor with the microbial membrane is fairly slow, This slow response may be due to the slow diffusion of ammonium ion from the sample solution to the surface of the PVC membrane across the microbial membrane.
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3.3. Effectiveness of microbial membrane against organic compounds As described in the section on response mechanisms, if the microbial membrane functions effectively, the present sensor will not be subject to interference from organic compounds. To examine effectiveness of the microbial membrane against organic compounds, the potential response of the ammonium ion sensor with and without the microbial membrane was examined for three standard solutions of ammonium ion (10⫺4, 10⫺3 and 10⫺2 mol l⫺1) and the same standard solutions containing 10⫺5 mol l⫺1 dodecytrimethylammonium ion (DOTA). DOTA was chosen as a typical organic compound found in waste water. The potentials of the sensor with and without the microbial membrane were measured for the above solutions and the results are shown in Fig. 4. A good linear relationship between the potential and the logarithmic concentration of ammonium ion was obtained for both sensors. However, the potential response of the sensor without the microbial membrane was shifted by 250 mV in a positive direction for the standard solution containing DOTA, compared to the standard solution without DOTA. In addition, the potential response of the sensor with the microbial membrane to the standard solution containing DOTA was nearly the same as for the standard solution without DOTA. This strongly suggests that DOTA was assimilated by the immobilized Trichosporon cutaneum in the microbial membrane when DOTA and ammonium ion diffused into the microbial membrane and that the interference from DOTA was eliminated. The assimilation rate of Trichosporon cutaneum for DOTA at the present
Fig. 4. Effectiveness of the microbial membrane against organic compound. Sample: standard ammonium ion solution. ⴰ The conventional sensor. • The present sensor with a microbial membrane. Sample: standard ammonium solution containing 10⫺5 mol l⫺1 dodecyltrimethyl ammonium ion. 왕 The conventional sensor. 왖 The present sensor with a microbial membrane.
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concentration would be predicted to be sufficiently faster than the diffusion rate of DOTA through the microbial membrane. The assimilation ability of the microbial membrane may be effective for other organic compounds judging from data on the BOD sensor fabricated with the same microbial membrane (Karube et al., 1977; Hikuma et al., 1979; Marty et al., 1997). 3.4. Correlation between the present sensor method and the Japanese Industrial Standard method for determination of ammonium ion in sewage samples The present sensor was applied to the determination of ammonium ion in sewage. The analytical results obtained by this method were correlated with data obtained by the Japanese Industrial Standard method (JIS: Indophenol method) (Japanese Industrial Standard, 1991). The correlation data are shown in Fig. 5. A regression line expressed by Y = 1.01X + 0.963x10⫺4 with a correlation factor of 0.999 between both methods was obtained if the result for the sample which contained ammonium ion at 10⫺2 mol l⫺1 is omitted. Fig. 5 indicates that the analytical results by the present sensor method are in good agreement with those by the JIS method for samples containing ammonium ion concentration of less than 5 × 10⫺3 mol l⫺1. However, the analytical result for the sample which contained about 1 × 10⫺2 mol l⫺1 as obtained by the present sensor method was higher than that by the JIS method by 20%. This discrepancy may arise from the fact that the activity of the microorganism in the microbial membrane is deactivated by these high levels of ammonium ion and, thus, organic compounds in waste water may interfere with the sensor. Such deactivation of microorganisms by inorganic ions is often observed in similar biosensors (Karube et al., 1977). The present sensor method is
Fig. 5. Correlation between the present method and Japanese Industrial Standard Method. Samples: Sewage waste water. Regression line: Y = 1.01X + 0.962 × 10⫺4. Correlation factor: r = 0.999.
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clearly practical for the determination of ammonium ion at the concentration less than 5 × 10⫺3 mol l⫺1 in sewage. 3.5. Long-term stability in sewage The long-term stability of the ammonium ion sensor with the microbial membrane, together with the conventional ammonium ion sensor without the microbial membrane, was investigated during analysis of sewage over a period of 130 days. Both sensors were immersed in the sewage sample during this period. The potential response of the sensor was measured for two standard solutions of 10⫺4 and 10⫺2 mol l⫺1 ammonium chloride at regular intervals, and the potential slope against the logarithmic concentration of ammonium ion was then obtained. As shown in Fig. 6, the potential slope of the ammonium ion sensor without the microbial membrane rapidly decreased after 5 days of use. However, the ammonium ion sensor with the microbial membrane maintained its initial performance with a near Nernst slope (56 mV/decade) over the entire 120 day period. This suggests that there was no deterioration in performance of this sensor during this period, even for the case of the sewage sample. We attribute this to the fact that the microbial membrane completely prevents organic compounds from coming into contact with the sensing PVC membrane. This experiments also shows that the use of the membrane, immobilized with the aerobic microorganism (Trichosporon cutaneum), significantly increases the life-time of the PVC membrane-based ammonium sensor.
Fig. 6. Life-time test of ammonium ion sensors with and without microbial membrane in sewage sample. -쐌- with microbial membrane ⴰ- without microbial membrane.
4. CONCLUSIONS We have developed a new type of PVC membranebased ammonium ion sensor with a microbial membrane, in which Trichosporon cutaneum is immobilized. This sensor proved to be durable over a long period of continuous use in sewage analysis. The effective elimination of interference from organic compounds was found to be due to the ability of the microorganism to assimilate organic compounds. As a result, compared with the conventional ammonium ion sensor without the microbial membrane, the lifetime of the present sensor in sewage was increased from 5 to 120 days. Furthermore, the determination of ammonium ion in the presence of 10⫺5 mol l⫺1 dodecytrimethylammonium ion could be carried out using the present sensor method. The present sensor was applicable to the determination of ammonium ion in sewage. A good correlation (r = 0.999) was obtained between the analytical results obtained by the present sensor method and those by the conventional method. We conclude that the present sensor method has advantages of simplicity, low cost and ease of maintenance for the direct continuous monitoring for ammonium ion. The basic concept, namely the elimination of interference from organic compounds by use of the microbial membrane may be applicable to other potentiometric PVC membrane-based sensors.
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