Journal of Biotechnology, 15 (1990) 255-266
255
Elsevier BIOTEC 00457
Microbial sensor Isao Karube Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
(Accepted 30 September 1989)
Introduction
Spectrophotometry and chromatography can be used for the determination of organic compounds, but they are not suitable for on-line measurement of these compounds. Electrochemical determination of such compounds has distinct advantages: for example, samples can be measured over a wide concentration range without pretreatment and do not need to be optically clear. Recently, m a n y biosensors have been developed for the determination of organic compounds. Enzyme sensors are highly specific for the substrate of interest, but the enzymes employed are generally expensive and unstable. Microbial sensors are composed of immobilized microorganisms and an electrochemical device and are suitable for the on-line determination of organic compounds. The microbial sensors developed by the author involve the assimilation of organic compounds by the microorganisms, change in respiration activity, or the production of electroactive metabolites; these being monitored directly by an electrochemical device. This paper describes several microbial sensors currently being developed by the authors.
BOD
sensor
The biochemical oxygen demand (BOD) is one of the most widely used and important tests in the measurement of organic pollution. The conventional BOD test requires a five-day incubation period, and values of the test results depend on the skill of an operator. Therefore, rapid and reproducible methods are desirable for the BOD test. A microbial sensor for BOD measurement utilizing metabolic ability of microorganisms was developed by the authors (Karube et al., 1987; H i k u m a et al., 1979a). For this purpose a yeast strain, Trichosporon cutaneum which was utilized for waste water treatment, was employed.
Correspondence to: I. Karube, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo 153, Japan.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
256 T. c u t a n e u m was immobilized in porous acetylcellulose m e m b r a n e by adsorption. A microorganism's immobilizing membrane was placed on a gas permeable Teflon membrane of Clark-type oxygen electrode, and covered with a dialysis m e m b r a n e to construct a microbial sensor. The measurement was attempted by flow analysis. The current at time zero was obtained with the buffer solution. This current shows endogeneous respiration level of the immobilized microorganisms. When the sample solution containing glucose and glutamic acid was injected into the system, organic compounds permeated through the porous m e m b r a n e and were assimilated by the immobilized microorganisms. Consumption of oxygen by the immobilized microorganisms began and caused a decrease in dissolved oxygen around the membranes. As a result, the current of the electrode decreased markedly with time until a steady state reached within 18 min. The steady state indicates the consumption of oxygen by microorganisms and that diffused from a sample solution to the m e m b r a n e were in equilibrium. The steady state current depended on BOD of the sample solution. Then the current of the microbial sensor finally returned to the initial level. The response time of the microbial sensor (time required for the current to reach steady state) also depended on the kind of sample solution. A linear relationship was observed between the current difference (between initial and steady state current) and the 5-day BOD of the standard solution below 60 mg 1-1. The minimum measurable BOD was 3 mg 1-1. The current was reproducible within +6% of the relative error when 40 mg 1-1 of the standard solution was employed. The standard deviation was BOD 1.2 mg 1-1 in 10 experiments. The microbial sensor was applied to the estimation of 5-day BOD for untreated waste waters from a bioplant. The 5-d BOD of the waste waters was determined by JIS method (Japanese Industrial Standard Committee, 1974). As shown in Fig. 1, good comparative results were obtained between BOD estimated by the microbial
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" ~ ~3
~ ~2
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0
I
1
I
2
I
3
I
4
5
BOD determined by JIS method (g ]") Fig. 1. Comparison between BOD determined by microbial sensor and BOD determination by JIS 5-day method.
Sample
Tap water
I I
I I
Flow line selector unit
Standard solution
I
'
Selector valve
Buffer tank
a
I
Sensorunff
I
O-lOOmV
I I
/u°,
I I1 . _ _ I ~.J Data --] processing
I IWaste
/ | |
I'-o.~v T- 7 II
c
=
~ l e
[
Microbial sensor
. . . .
Fig. 2. Schematic diagram of continual measuring system for BOD. a, pump; b, filter; c, incubator; f, flow meter; e, air pump.
Sampling unit
Waste
Sample
Rinsate
Selector controller
I
258 sensor and those determined by JIS method. Regression coefficient was 1.2 in 17 experiments and the ratios (BOD estimated by the microbial sensor vs. 5-d BOD determined by JIS method) were in the range from 0.85 to 1.36. This variation might have been caused from change in composition of organic compounds in waste waters. Therefore, the effect of various pure compounds on BOD estimated by the microbial sensor was examined. The sensor showed low BOD values compared with the 5-d BOD when lactose and soluble starch were employed for experiments. This might be caused from the slow decomposition rate of these compounds by the immobilized yeasts. On the other hand, the sensor showed high BOD values compared with the 5-d BOD when acetic acid and ethyl alcohol were employed. These results suggested the oxidation rate of acetate and ethyl alcohol to be faster than that of some standard substrates such as glucose and glutamic acid. No large difference was observed between the BOD determined by both methods when other substrates were employed for experiments. Therefore, waste waters containing substrates such as soluble starch showed low BOD values and those containing substrates such as acetic acid and alcohols showed high BOD values. Stable responses to the standard solution (BOD 20 mg 1-1) were observed for more than 17 d (400 tests). Fluctuations of the current and base line were within 20% and 15% respectively for 17 d. The microbial sensor could be used for a long time for the estimation of BOD. The microbial sensors for BOD measurement have been already commercialized in Japan (Fig. 2). The measurements taken using this sensor usually employ a flow cell system. The measurement is based on either steady state method, by feeding sample solution continuously in the flow cell or pulse method, (flow injection analysis) based on injecting appropriate amounts of sample solution into the flow line. In conclusion, the microbial sensor using immobilized yeasts appears quite promising and very attractive for the estimation of the 5-day BOD of industrial waste waters.
Ethanol sensor
On-line measurements of ethanol concentrations are required in various industries. Many reports on applications of enzyme electrodes have been published for the determination of alcohols. However, enzymes are generally expensive and unstable. It is well known that many microorganisms utilize alcohol as carbon. Therefore, it is possible to construct a microbial sensor for alcohols using immobilized microorganisms and an oxygen electrode. A microbial sensor consisting of immobilized yeasts, a gas permeable Teflon membrane, and an oxygen electrode was prepared for the determination of ethanol (Hikuma et al., 1979b). A linear relationship was observed between the current decrease and the concentration of ethanol below 22.5 mg 1-1 by pulse methods. The minimum ethanol concentration for the determination was 2 mg 1-1. The reproducibility of the current
259 difference was examined using the same sample. The current difference was reproducible within 6% of the relative error when a sample solution containing 16.5 mg 1-~ of ethanol was employed. The standard deviation was 0.5 mg 1-1 in 40 experiments. The selectivity of the microbial sensor for ethanol was examined. The sensor did not respond to volatile compounds such as methanol, formic acid, acetic acid, propionic acid, and other nutrients for microorganisms such as carbohydrates, amino acids, and ions. As the microbes were covered with a gas permeable membrane, only volatile compounds can penetrate through the membrane. However, an ethanol-utilizing yeast, T. brassicae, did not utilize methanol. The selectivity of the microbial sensor for ethanol was satisfactory. The microbial sensor for ethanol was applied to a complex medium used for yeast growth. The concentration of ethanol was determined by the microbial sensor and by gas chromatography. Satisfactory comparative results were obtained between them. The correlation coefficient was 0.98 with 20 experiments. Yeasts whole cells did not affect the electrochemical determination of ethanol. The reusability of the microbial sensor for ethanol was examined. Ethanol solutions (from 5.5 to 22.3 mg 1-1) were used for long-term stability testing of the sensor. The current output of the sensor was almost constant for more than three weeks and 2100 assays. Therefore, the yeasts in the electrode were viable for a long time for assay of ethanol.
Acetic acid sensor
On-line measurement of acetic acid concentrations is required in various industries. Trichosporon brassicae, which was utilized in an ethanol sensor, is also known to utilize acetic acid as a carbon source. By keeping the p H of buffer solution at lower than p K of acetic acid, vaporized acetic acid can be determined by using a microbial sensor constructed for an ethanol measurement (Hikuma et al., 1979c). The measurements were attempted by flow analyses. Within 3 min, the m a x i m u m response for a sample was obtained. The total time required for an assay was 15 min. The calibration graphs obtained showed linear relationships between the current decrease and the concentration of acetic acid up to 72 mg 1-~. The minimum concentration for determination was 5 mg of acetic acid per liter. The reproducibility of the current difference was examined using the same sample. The current difference was reproducible within 6% for an acetic acid sample containing 54 mg l-1. The standard deviation was 1.6 mg 1-1 in 20 experiments. The selectivity of the microbial sensor for acetic acid was examined. The sensor did not respond to non-volatile compounds such as glucose and phosphate ions. As the microbial sensor was covered with a gas-permeable membrane, only volatile compounds could penetrate through the membrane. The response to organic compounds depends on the assimilation of the immobilized microorganisms, T. brassi-
260 cae, which utilizes acetic acid, n-butyric acid and ethanol. Fortunately, the latter
three compounds are not generally present in this medium. The microbial sensor for acetic acid was applied to a medium containing glutamic acid. The concentration of acetic acid was determined by the microbial sensor and by a gas chromatographic method. Good agreement was obtained; the regression coefficient was 1.04 for 26 experiments. Whole cells in the medium did not affect the electrochemical determination of acetic acid. The long-term stability of the microbial sensor was examined for acetic acid solution (72 mg 1-1). The current output (0.29-0.25/~A) of the sensor was constant (within +10% of the original values) for more than 3 weeks and 1500 assays. Therefore, the yeasts in the electrode survived remarkably well. In conclusion, the microbial sensor appears to be quite promising and very attractive for the determination of acetic acid in reaction mixtures.
Micro-ethanol sensor
For the on-line monitoring of industrial processes, the small and selective ethanol sensor is appreciable. A micro-alcohol sensor consisting of immobilized acetic acid bacteria, a gas permeable membrane and an I S F E T was developed by the authors (Kitagawa et al., 1987). Acetic acid bacteria have been widely used in the industries to make vinegar and ascorbic acid by the oxidative degradation. In acetic acid processes, both alcohol dehydrogenase ( A D H ) and aldehyde dehydrogenase ( A L D H ) are involved in oxidization of ethanol to acetic acid via acetaldehyde. When acetic acid bacteria are immobilized on an ISFET, ethanol is oxidized to acetic acid by those bacteria, causing a p H change to occur at the surface of ISFET. Therefore, ethanol concentration can be measured by combining an I S F E T and acetic acid bacteria. Acetobacter aceti I A M 1802 was used for the micro-alcohol sensor. The microorganisms were immobilized on the gate surface of ISFET, in calcium alginate gel. The ISFET-immobilized microorganisms and a wire-like Ag-AgC1 electrode were placed in a small shell (6 x 3 x 22 mm) that had a gas permeable membrane (a porous Teflon membrane, 0.5/zm pore size) fitted to the side. The inside of the shell was filled with inner buffer solution (5 m M Tris-HC1 buffer containing 0.1 M CaC12, p H 7.0). The system consisted of a thermostatic circulating jacketed vessel (3 ml volume), the microbial-FET alcohol sensor, the circuit for measurement of the gate output voltage (Vg), an electrometer and a recorder. The initial rate change of gate voltage with time, d V / d t , was plotted against the logarithmic value of the ethanol concentration. The minimum detectable response to ethanol was obtained at approximately 0.1 mM. A linear relationship was observed over the range between 3-70 m M (Fig. 3). The response of the micro-alcohol sensor was stable over a wide p H range (pH 2-12), however, at pHs lower than 6, the sensor responded to acetic acid. Volatile organic acids such as acetic acid do not dissociate in lower p H solution, and
261 50
40 Ic..
•~ 30 E E v
j
2O o') "0
10
0 0,1
I
I
1,0
10
Ethanol concentration
i00
(mM)
Fig. 3. Calibration curve for ethanol.
penetrate through the gas permeable membrane, at p H s higher than 6, on the other hand, the volatile organic acids dissociate to the individual ions, they did not interfere with the measurement of ethanol by sensor. The sensor was stable for 15 h, at 15 o C. The sensor showed usefulness for the determination of ethanol, especially in terms of its good selectivity and potential for miniaturization, and it is expected to be integrated into a multi-functional sensor capable of simultaneously determining substrates contained in a solution having a complex composition.
Carbon dioxide sensor
The determination of carbon dioxide is very important in some industrial processes, in environmental and clinical analyses. The potentiometric p C O 2 device has been widely utilized; however, major problems are associated with the potentiometric method of detection. Therefore, the authors have developed a novel microbial CO 2 sensor based on amperometry (Suzuki et al., 1987, 1988). The chemoautotrophic bacterium, which can grow with only carbonate as the carbon-source, was used in this sensor. This bacterium was isolated from soil obtained in Akita prefecture, Japan, and n a m e d S-17 (provided by the Fermentation Research Institute, Japan). S-17 was autotrophically incubated under aerobic conditions at 30 ° C for approximately one month in an inorganic medium and used for the microbial sensor. Bacterial cells immobilized on a cellulose nitrate m e m b r a n e (pore size 0.45 #m) were placed in the vicinity of the cathode of a Galvanic-type oxygen electrode. The
262
Immobilized bacteria
Buffer solution
membrane
Dialysis membrane
Gas permeable membrane
Fig. 4. Pfinciple ofthemicrobialsensorfor CO2 measurement. cathode and the immobilized cells were then totally covered with a gas-permeable membrane, polytetrafluoroethylene (PTFE). When a sample solution containing COz, K z C O 3 was injected into the external buffer solution, CO 2 permeated through the gas permeable membrane, and was assimilated by the bacteria. The respiratory rate of the bacteria consequently became elevated, giving rise to the consumption of oxygen (Fig. 4). The current decreased rapidly and reached m a x i m u m response in 2 - 5 min after injection of KzCO 3 followed by the gradual increase of the current due to the escape of CO 2 to the air. The CO 2 concentration was determined by the microbial sensor and by a conventional potentiometric p C O 2 electrode. The linear range of the sensor was found to be below 200 p p m CO 2. The upper limit of the linear range was determined by the saturation of CO 2 in the buffer solution and not by considering that current fluctuation resulting from experimental errors is within 0.5 /~A; our novel sensor could easily detect a variation of 5 p p m CO 2 (200/LM K2CO3) in the lower concentration range. Selectivity of the microbiological sensor was also examined. Various aqueous solutions of volatile compounds such as acetic acid, citric acid, formic acid, ethanol, butanol were added to the sensor system. Although the sensor responded slightly (37% of the response to CO2) to acetic acid, all the other compounds produced no observable response. Stability of the sensor was more than three weeks.
Micro-carbon dioxide sensor
The detection of carbon dioxide (CO2) is very important in m a n y industrial processes and clinical analyses. In previous studies, we fabricated bacterial CO 2 sensors using a conventional Galvanic oxygen electrode and autotrophic bacteria. The use of microelectrodes enables the miniaturization of the CO 2 sensor, conse-
263
Sensitive area /
Anode
/Cathode -A'
Gas-permeable membrane ,///
Immobilized bacteria
J
~
~
/ /
\
Hydrophobic insulator
Electrolyteimpregnated gel Pads
A-A' cross section
2~ Fig. 5. St~cture of ~e CO2 sensor using a ~crooxygen d~trode.
quently allows the measurement using only minute amount of samples and also the integration of several biosensors in one chip. The major objective of our research was to develop a disposable microbial CO 2 sensor using the miniature oxygen electrode and bacteria (Suzuki et al., 1989). The CO 2 sensor structure is shown in Fig. 5. The electrode, formed on a 2 × 15 m m silicon chip, has a V-shaped groove formed by anisotropic etching of silicon, a gold cathode and a gold anode. The electrodes were formed over a SiO 2 layer that electrically insulates them. Anisotropically etched grooves were formed only in the anode. The cathode was not etched, but, instead, a small recess was formed b y the negative photoresist to hold the electrolyte solution. The rectangular resist pattern also determined sensitive area. The sensitive area was 0.2 x 2 mm. Calcium alginate gel containing a 0.1 M potassium chloride (KC1) aqueous solution as the electrolyte fills the groove and is covered by the gas-permeable membrane. Only the pad areas of the two gold electrodes, where a constant voltage was applied and the oxygen reduction current was measured, were exposed. The opposite side of the wafer was coated with a hydrophobic insulator. Without the insulator, the device is affected b y noise and the generated current outputs differ from device to device (from nA to/~A). The ratio of the areas of the cathode to the anode was about 1 : 2 . Oxygen concentration is measured by the oxygen electrode as the oxygen reduction current with a constant voltage between the cathode and anode. The autotrophic bacteria were immobilized on the sensitive area of the oxygen electrode by incorporating them in a sodium alginate gel matrix, and the gas-permeable m e m b r a n e was reformed over the bacteria-immobilized gel. If a sample solution containing the CO 2 is added to the buffer solution in which the sensitive area of the sensor is immersed, the CO 2 permeates the membrane,
264
0.3
3 (1) 03
02 /X"
t_
t._
0.1
0
4
8
CO 2 concentration
12 (%)
Fig. 6. Calibration curve of the microbial sensor for CO2 measurement. reaches the bacteria, and is assimilated. Bacterial respiration increases and they consume more oxygen. The change in oxygen concentration inside the gaspermeable membrane is detected as an oxygen reduction current on the cathode electrode, and the CO 2 concentration can be determined indirectly. The response time of the sensor was from 2 to 3 min, and seems to mainly depend on the oxygen electrode rather than the diffusion of the CO 2 through the bacteria immobilized in the gel. The 90% response time of the oxygen electrode was 30 s to 2 min, when the photoresist was used for the gas-permeable membrane. Fig. 6 shows a CO 2 sensor calibration curve at 30°C. The CO 2 was supplied by acidification of N a H C O 3 and the N a H C O 3 concentration was related to CO 2 concentrations. The lowest detection limit was 0.5 m M N a H C O 3 within the margin of the noise amplitude. A linear relationship was observed at N a H C O 3 concentrations between 0.5 mM and 3.5 m M at 30 ° C, p H 5.5. Above 3.5 raM, there were no significant increases in response. The selectivity of the CO 2 sensor was examined. Because the surface of the sensitive area is covered by gas-permeable membrane, ions cannot permeate into the bacteria-immobilized membrane or inside of the oxygen electrode. Only gas can be assimilated by the bacteria. Aqueous solutions of sodium acetate, trisodium citrate, methanol, ethanol, 1-propanol, 1-butanol, and urea were added to the system. Organic acids were added as their sodium salts so the p H of the solution would not be affected. The sensor responded to sodium acetate and 1-propanol. The responses to these agents were less than 20% that to N a H C O 3. Other compounds were not responded. Responses to acetate and 1-propanol are possibly due to the volatility of the compounds and they were used by the bacteria. The sensor can be used more than 10 times.
265
References Karube, I., Matsunaga, T., Mitsuda, S. and Suzuki, S. (1977) Microbial electrode BOD sensors. Biotechnol. Bioeng. 19, 1535-1547. Karube, I., (1987) Microbiosensor based on silicon fabrication technology. In: (Turner, A.P.F., Karube, I. and Wilson, G.S. (Eds.), Biosensors-Fundamentals and Applications. Oxford University Press, Oxford, pp. 471-480. Kitagawa, Y., Tamiya, E. and Karube, I. (1987) Microbial-FET alcohol sensor. Anal. Lett. 20, 81-96. Hikuma, M., Kubo, T., Yasuda, T., Karube, I. and Suzuki, S. (1979a) Microbial electrode sensor for alcohols. Biotechnol. Bioeng. 21, 1845-1853. Hikuma, M., Kubo, T., Yasuda, T., Karube, I. and Suzuki, S. (1979b) Amperometric determination of acetic acid with immobilized Trichosporon brassicae. Anal. Chim. Acta 109, 33-38. Hikuma, M., Suzuki, H., Yasuda, T., Karube, I. and Suzuki, S. (1979c) Amperometric estimation of BOD by using living immobilized yeasts. Eur. J. Appl. Microb. Biotechnol. 8, 289-297. Suzuki, H., Kojima, N., Sugama, A., Takei, F., Ikegami, K., Tamiya, E. and Karube, I. (1989) Fabrication of a microbial carbon dioxide sensor using semiconductor fabrication techniques. Electroanalysis 1, 305-309. Suzuki, H., Tamiya, E. and Karube, I. (1987) An amperometric sensor for carbon dioxide. Anal. Chim. Acta 199, 85-91. Suzuki, H., Tamiya, E., Karube, I. and Ooshima, T. (1988) Carbon dioxide sensor using thermophilic bacteria. Anal. Lett. 21, 1323-1336.
255
Elsevier BIOTEC 00457
Microbial sensor Isao Karube Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
(Accepted 30 September 1989)
Introduction
Spectrophotometry and chromatography can be used for the determination of organic compounds, but they are not suitable for on-line measurement of these compounds. Electrochemical determination of such compounds has distinct advantages: for example, samples can be measured over a wide concentration range without pretreatment and do not need to be optically clear. Recently, m a n y biosensors have been developed for the determination of organic compounds. Enzyme sensors are highly specific for the substrate of interest, but the enzymes employed are generally expensive and unstable. Microbial sensors are composed of immobilized microorganisms and an electrochemical device and are suitable for the on-line determination of organic compounds. The microbial sensors developed by the author involve the assimilation of organic compounds by the microorganisms, change in respiration activity, or the production of electroactive metabolites; these being monitored directly by an electrochemical device. This paper describes several microbial sensors currently being developed by the authors.
BOD
sensor
The biochemical oxygen demand (BOD) is one of the most widely used and important tests in the measurement of organic pollution. The conventional BOD test requires a five-day incubation period, and values of the test results depend on the skill of an operator. Therefore, rapid and reproducible methods are desirable for the BOD test. A microbial sensor for BOD measurement utilizing metabolic ability of microorganisms was developed by the authors (Karube et al., 1987; H i k u m a et al., 1979a). For this purpose a yeast strain, Trichosporon cutaneum which was utilized for waste water treatment, was employed.
Correspondence to: I. Karube, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo 153, Japan.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
256 T. c u t a n e u m was immobilized in porous acetylcellulose m e m b r a n e by adsorption. A microorganism's immobilizing membrane was placed on a gas permeable Teflon membrane of Clark-type oxygen electrode, and covered with a dialysis m e m b r a n e to construct a microbial sensor. The measurement was attempted by flow analysis. The current at time zero was obtained with the buffer solution. This current shows endogeneous respiration level of the immobilized microorganisms. When the sample solution containing glucose and glutamic acid was injected into the system, organic compounds permeated through the porous m e m b r a n e and were assimilated by the immobilized microorganisms. Consumption of oxygen by the immobilized microorganisms began and caused a decrease in dissolved oxygen around the membranes. As a result, the current of the electrode decreased markedly with time until a steady state reached within 18 min. The steady state indicates the consumption of oxygen by microorganisms and that diffused from a sample solution to the m e m b r a n e were in equilibrium. The steady state current depended on BOD of the sample solution. Then the current of the microbial sensor finally returned to the initial level. The response time of the microbial sensor (time required for the current to reach steady state) also depended on the kind of sample solution. A linear relationship was observed between the current difference (between initial and steady state current) and the 5-day BOD of the standard solution below 60 mg 1-1. The minimum measurable BOD was 3 mg 1-1. The current was reproducible within +6% of the relative error when 40 mg 1-1 of the standard solution was employed. The standard deviation was BOD 1.2 mg 1-1 in 10 experiments. The microbial sensor was applied to the estimation of 5-day BOD for untreated waste waters from a bioplant. The 5-d BOD of the waste waters was determined by JIS method (Japanese Industrial Standard Committee, 1974). As shown in Fig. 1, good comparative results were obtained between BOD estimated by the microbial
O3 v
n
~-4 o c/)
" ~ ~3
~ ~2
"0
u-
0
I
1
I
2
I
3
I
4
5
BOD determined by JIS method (g ]") Fig. 1. Comparison between BOD determined by microbial sensor and BOD determination by JIS 5-day method.
Sample
Tap water
I I
I I
Flow line selector unit
Standard solution
I
'
Selector valve
Buffer tank
a
I
Sensorunff
I
O-lOOmV
I I
/u°,
I I1 . _ _ I ~.J Data --] processing
I IWaste
/ | |
I'-o.~v T- 7 II
c
=
~ l e
[
Microbial sensor
. . . .
Fig. 2. Schematic diagram of continual measuring system for BOD. a, pump; b, filter; c, incubator; f, flow meter; e, air pump.
Sampling unit
Waste
Sample
Rinsate
Selector controller
I
258 sensor and those determined by JIS method. Regression coefficient was 1.2 in 17 experiments and the ratios (BOD estimated by the microbial sensor vs. 5-d BOD determined by JIS method) were in the range from 0.85 to 1.36. This variation might have been caused from change in composition of organic compounds in waste waters. Therefore, the effect of various pure compounds on BOD estimated by the microbial sensor was examined. The sensor showed low BOD values compared with the 5-d BOD when lactose and soluble starch were employed for experiments. This might be caused from the slow decomposition rate of these compounds by the immobilized yeasts. On the other hand, the sensor showed high BOD values compared with the 5-d BOD when acetic acid and ethyl alcohol were employed. These results suggested the oxidation rate of acetate and ethyl alcohol to be faster than that of some standard substrates such as glucose and glutamic acid. No large difference was observed between the BOD determined by both methods when other substrates were employed for experiments. Therefore, waste waters containing substrates such as soluble starch showed low BOD values and those containing substrates such as acetic acid and alcohols showed high BOD values. Stable responses to the standard solution (BOD 20 mg 1-1) were observed for more than 17 d (400 tests). Fluctuations of the current and base line were within 20% and 15% respectively for 17 d. The microbial sensor could be used for a long time for the estimation of BOD. The microbial sensors for BOD measurement have been already commercialized in Japan (Fig. 2). The measurements taken using this sensor usually employ a flow cell system. The measurement is based on either steady state method, by feeding sample solution continuously in the flow cell or pulse method, (flow injection analysis) based on injecting appropriate amounts of sample solution into the flow line. In conclusion, the microbial sensor using immobilized yeasts appears quite promising and very attractive for the estimation of the 5-day BOD of industrial waste waters.
Ethanol sensor
On-line measurements of ethanol concentrations are required in various industries. Many reports on applications of enzyme electrodes have been published for the determination of alcohols. However, enzymes are generally expensive and unstable. It is well known that many microorganisms utilize alcohol as carbon. Therefore, it is possible to construct a microbial sensor for alcohols using immobilized microorganisms and an oxygen electrode. A microbial sensor consisting of immobilized yeasts, a gas permeable Teflon membrane, and an oxygen electrode was prepared for the determination of ethanol (Hikuma et al., 1979b). A linear relationship was observed between the current decrease and the concentration of ethanol below 22.5 mg 1-1 by pulse methods. The minimum ethanol concentration for the determination was 2 mg 1-1. The reproducibility of the current
259 difference was examined using the same sample. The current difference was reproducible within 6% of the relative error when a sample solution containing 16.5 mg 1-~ of ethanol was employed. The standard deviation was 0.5 mg 1-1 in 40 experiments. The selectivity of the microbial sensor for ethanol was examined. The sensor did not respond to volatile compounds such as methanol, formic acid, acetic acid, propionic acid, and other nutrients for microorganisms such as carbohydrates, amino acids, and ions. As the microbes were covered with a gas permeable membrane, only volatile compounds can penetrate through the membrane. However, an ethanol-utilizing yeast, T. brassicae, did not utilize methanol. The selectivity of the microbial sensor for ethanol was satisfactory. The microbial sensor for ethanol was applied to a complex medium used for yeast growth. The concentration of ethanol was determined by the microbial sensor and by gas chromatography. Satisfactory comparative results were obtained between them. The correlation coefficient was 0.98 with 20 experiments. Yeasts whole cells did not affect the electrochemical determination of ethanol. The reusability of the microbial sensor for ethanol was examined. Ethanol solutions (from 5.5 to 22.3 mg 1-1) were used for long-term stability testing of the sensor. The current output of the sensor was almost constant for more than three weeks and 2100 assays. Therefore, the yeasts in the electrode were viable for a long time for assay of ethanol.
Acetic acid sensor
On-line measurement of acetic acid concentrations is required in various industries. Trichosporon brassicae, which was utilized in an ethanol sensor, is also known to utilize acetic acid as a carbon source. By keeping the p H of buffer solution at lower than p K of acetic acid, vaporized acetic acid can be determined by using a microbial sensor constructed for an ethanol measurement (Hikuma et al., 1979c). The measurements were attempted by flow analyses. Within 3 min, the m a x i m u m response for a sample was obtained. The total time required for an assay was 15 min. The calibration graphs obtained showed linear relationships between the current decrease and the concentration of acetic acid up to 72 mg 1-~. The minimum concentration for determination was 5 mg of acetic acid per liter. The reproducibility of the current difference was examined using the same sample. The current difference was reproducible within 6% for an acetic acid sample containing 54 mg l-1. The standard deviation was 1.6 mg 1-1 in 20 experiments. The selectivity of the microbial sensor for acetic acid was examined. The sensor did not respond to non-volatile compounds such as glucose and phosphate ions. As the microbial sensor was covered with a gas-permeable membrane, only volatile compounds could penetrate through the membrane. The response to organic compounds depends on the assimilation of the immobilized microorganisms, T. brassi-
260 cae, which utilizes acetic acid, n-butyric acid and ethanol. Fortunately, the latter
three compounds are not generally present in this medium. The microbial sensor for acetic acid was applied to a medium containing glutamic acid. The concentration of acetic acid was determined by the microbial sensor and by a gas chromatographic method. Good agreement was obtained; the regression coefficient was 1.04 for 26 experiments. Whole cells in the medium did not affect the electrochemical determination of acetic acid. The long-term stability of the microbial sensor was examined for acetic acid solution (72 mg 1-1). The current output (0.29-0.25/~A) of the sensor was constant (within +10% of the original values) for more than 3 weeks and 1500 assays. Therefore, the yeasts in the electrode survived remarkably well. In conclusion, the microbial sensor appears to be quite promising and very attractive for the determination of acetic acid in reaction mixtures.
Micro-ethanol sensor
For the on-line monitoring of industrial processes, the small and selective ethanol sensor is appreciable. A micro-alcohol sensor consisting of immobilized acetic acid bacteria, a gas permeable membrane and an I S F E T was developed by the authors (Kitagawa et al., 1987). Acetic acid bacteria have been widely used in the industries to make vinegar and ascorbic acid by the oxidative degradation. In acetic acid processes, both alcohol dehydrogenase ( A D H ) and aldehyde dehydrogenase ( A L D H ) are involved in oxidization of ethanol to acetic acid via acetaldehyde. When acetic acid bacteria are immobilized on an ISFET, ethanol is oxidized to acetic acid by those bacteria, causing a p H change to occur at the surface of ISFET. Therefore, ethanol concentration can be measured by combining an I S F E T and acetic acid bacteria. Acetobacter aceti I A M 1802 was used for the micro-alcohol sensor. The microorganisms were immobilized on the gate surface of ISFET, in calcium alginate gel. The ISFET-immobilized microorganisms and a wire-like Ag-AgC1 electrode were placed in a small shell (6 x 3 x 22 mm) that had a gas permeable membrane (a porous Teflon membrane, 0.5/zm pore size) fitted to the side. The inside of the shell was filled with inner buffer solution (5 m M Tris-HC1 buffer containing 0.1 M CaC12, p H 7.0). The system consisted of a thermostatic circulating jacketed vessel (3 ml volume), the microbial-FET alcohol sensor, the circuit for measurement of the gate output voltage (Vg), an electrometer and a recorder. The initial rate change of gate voltage with time, d V / d t , was plotted against the logarithmic value of the ethanol concentration. The minimum detectable response to ethanol was obtained at approximately 0.1 mM. A linear relationship was observed over the range between 3-70 m M (Fig. 3). The response of the micro-alcohol sensor was stable over a wide p H range (pH 2-12), however, at pHs lower than 6, the sensor responded to acetic acid. Volatile organic acids such as acetic acid do not dissociate in lower p H solution, and
261 50
40 Ic..
•~ 30 E E v
j
2O o') "0
10
0 0,1
I
I
1,0
10
Ethanol concentration
i00
(mM)
Fig. 3. Calibration curve for ethanol.
penetrate through the gas permeable membrane, at p H s higher than 6, on the other hand, the volatile organic acids dissociate to the individual ions, they did not interfere with the measurement of ethanol by sensor. The sensor was stable for 15 h, at 15 o C. The sensor showed usefulness for the determination of ethanol, especially in terms of its good selectivity and potential for miniaturization, and it is expected to be integrated into a multi-functional sensor capable of simultaneously determining substrates contained in a solution having a complex composition.
Carbon dioxide sensor
The determination of carbon dioxide is very important in some industrial processes, in environmental and clinical analyses. The potentiometric p C O 2 device has been widely utilized; however, major problems are associated with the potentiometric method of detection. Therefore, the authors have developed a novel microbial CO 2 sensor based on amperometry (Suzuki et al., 1987, 1988). The chemoautotrophic bacterium, which can grow with only carbonate as the carbon-source, was used in this sensor. This bacterium was isolated from soil obtained in Akita prefecture, Japan, and n a m e d S-17 (provided by the Fermentation Research Institute, Japan). S-17 was autotrophically incubated under aerobic conditions at 30 ° C for approximately one month in an inorganic medium and used for the microbial sensor. Bacterial cells immobilized on a cellulose nitrate m e m b r a n e (pore size 0.45 #m) were placed in the vicinity of the cathode of a Galvanic-type oxygen electrode. The
262
Immobilized bacteria
Buffer solution
membrane
Dialysis membrane
Gas permeable membrane
Fig. 4. Pfinciple ofthemicrobialsensorfor CO2 measurement. cathode and the immobilized cells were then totally covered with a gas-permeable membrane, polytetrafluoroethylene (PTFE). When a sample solution containing COz, K z C O 3 was injected into the external buffer solution, CO 2 permeated through the gas permeable membrane, and was assimilated by the bacteria. The respiratory rate of the bacteria consequently became elevated, giving rise to the consumption of oxygen (Fig. 4). The current decreased rapidly and reached m a x i m u m response in 2 - 5 min after injection of KzCO 3 followed by the gradual increase of the current due to the escape of CO 2 to the air. The CO 2 concentration was determined by the microbial sensor and by a conventional potentiometric p C O 2 electrode. The linear range of the sensor was found to be below 200 p p m CO 2. The upper limit of the linear range was determined by the saturation of CO 2 in the buffer solution and not by considering that current fluctuation resulting from experimental errors is within 0.5 /~A; our novel sensor could easily detect a variation of 5 p p m CO 2 (200/LM K2CO3) in the lower concentration range. Selectivity of the microbiological sensor was also examined. Various aqueous solutions of volatile compounds such as acetic acid, citric acid, formic acid, ethanol, butanol were added to the sensor system. Although the sensor responded slightly (37% of the response to CO2) to acetic acid, all the other compounds produced no observable response. Stability of the sensor was more than three weeks.
Micro-carbon dioxide sensor
The detection of carbon dioxide (CO2) is very important in m a n y industrial processes and clinical analyses. In previous studies, we fabricated bacterial CO 2 sensors using a conventional Galvanic oxygen electrode and autotrophic bacteria. The use of microelectrodes enables the miniaturization of the CO 2 sensor, conse-
263
Sensitive area /
Anode
/Cathode -A'
Gas-permeable membrane ,///
Immobilized bacteria
J
~
~
/ /
\
Hydrophobic insulator
Electrolyteimpregnated gel Pads
A-A' cross section
2~ Fig. 5. St~cture of ~e CO2 sensor using a ~crooxygen d~trode.
quently allows the measurement using only minute amount of samples and also the integration of several biosensors in one chip. The major objective of our research was to develop a disposable microbial CO 2 sensor using the miniature oxygen electrode and bacteria (Suzuki et al., 1989). The CO 2 sensor structure is shown in Fig. 5. The electrode, formed on a 2 × 15 m m silicon chip, has a V-shaped groove formed by anisotropic etching of silicon, a gold cathode and a gold anode. The electrodes were formed over a SiO 2 layer that electrically insulates them. Anisotropically etched grooves were formed only in the anode. The cathode was not etched, but, instead, a small recess was formed b y the negative photoresist to hold the electrolyte solution. The rectangular resist pattern also determined sensitive area. The sensitive area was 0.2 x 2 mm. Calcium alginate gel containing a 0.1 M potassium chloride (KC1) aqueous solution as the electrolyte fills the groove and is covered by the gas-permeable membrane. Only the pad areas of the two gold electrodes, where a constant voltage was applied and the oxygen reduction current was measured, were exposed. The opposite side of the wafer was coated with a hydrophobic insulator. Without the insulator, the device is affected b y noise and the generated current outputs differ from device to device (from nA to/~A). The ratio of the areas of the cathode to the anode was about 1 : 2 . Oxygen concentration is measured by the oxygen electrode as the oxygen reduction current with a constant voltage between the cathode and anode. The autotrophic bacteria were immobilized on the sensitive area of the oxygen electrode by incorporating them in a sodium alginate gel matrix, and the gas-permeable m e m b r a n e was reformed over the bacteria-immobilized gel. If a sample solution containing the CO 2 is added to the buffer solution in which the sensitive area of the sensor is immersed, the CO 2 permeates the membrane,
264
0.3
3 (1) 03
02 /X"
t_
t._
0.1
0
4
8
CO 2 concentration
12 (%)
Fig. 6. Calibration curve of the microbial sensor for CO2 measurement. reaches the bacteria, and is assimilated. Bacterial respiration increases and they consume more oxygen. The change in oxygen concentration inside the gaspermeable membrane is detected as an oxygen reduction current on the cathode electrode, and the CO 2 concentration can be determined indirectly. The response time of the sensor was from 2 to 3 min, and seems to mainly depend on the oxygen electrode rather than the diffusion of the CO 2 through the bacteria immobilized in the gel. The 90% response time of the oxygen electrode was 30 s to 2 min, when the photoresist was used for the gas-permeable membrane. Fig. 6 shows a CO 2 sensor calibration curve at 30°C. The CO 2 was supplied by acidification of N a H C O 3 and the N a H C O 3 concentration was related to CO 2 concentrations. The lowest detection limit was 0.5 m M N a H C O 3 within the margin of the noise amplitude. A linear relationship was observed at N a H C O 3 concentrations between 0.5 mM and 3.5 m M at 30 ° C, p H 5.5. Above 3.5 raM, there were no significant increases in response. The selectivity of the CO 2 sensor was examined. Because the surface of the sensitive area is covered by gas-permeable membrane, ions cannot permeate into the bacteria-immobilized membrane or inside of the oxygen electrode. Only gas can be assimilated by the bacteria. Aqueous solutions of sodium acetate, trisodium citrate, methanol, ethanol, 1-propanol, 1-butanol, and urea were added to the system. Organic acids were added as their sodium salts so the p H of the solution would not be affected. The sensor responded to sodium acetate and 1-propanol. The responses to these agents were less than 20% that to N a H C O 3. Other compounds were not responded. Responses to acetate and 1-propanol are possibly due to the volatility of the compounds and they were used by the bacteria. The sensor can be used more than 10 times.
265
References Karube, I., Matsunaga, T., Mitsuda, S. and Suzuki, S. (1977) Microbial electrode BOD sensors. Biotechnol. Bioeng. 19, 1535-1547. Karube, I., (1987) Microbiosensor based on silicon fabrication technology. In: (Turner, A.P.F., Karube, I. and Wilson, G.S. (Eds.), Biosensors-Fundamentals and Applications. Oxford University Press, Oxford, pp. 471-480. Kitagawa, Y., Tamiya, E. and Karube, I. (1987) Microbial-FET alcohol sensor. Anal. Lett. 20, 81-96. Hikuma, M., Kubo, T., Yasuda, T., Karube, I. and Suzuki, S. (1979a) Microbial electrode sensor for alcohols. Biotechnol. Bioeng. 21, 1845-1853. Hikuma, M., Kubo, T., Yasuda, T., Karube, I. and Suzuki, S. (1979b) Amperometric determination of acetic acid with immobilized Trichosporon brassicae. Anal. Chim. Acta 109, 33-38. Hikuma, M., Suzuki, H., Yasuda, T., Karube, I. and Suzuki, S. (1979c) Amperometric estimation of BOD by using living immobilized yeasts. Eur. J. Appl. Microb. Biotechnol. 8, 289-297. Suzuki, H., Kojima, N., Sugama, A., Takei, F., Ikegami, K., Tamiya, E. and Karube, I. (1989) Fabrication of a microbial carbon dioxide sensor using semiconductor fabrication techniques. Electroanalysis 1, 305-309. Suzuki, H., Tamiya, E. and Karube, I. (1987) An amperometric sensor for carbon dioxide. Anal. Chim. Acta 199, 85-91. Suzuki, H., Tamiya, E., Karube, I. and Ooshima, T. (1988) Carbon dioxide sensor using thermophilic bacteria. Anal. Lett. 21, 1323-1336.
