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New potentiometric microbial biosensor for ethanol determination in alcoholic beverages
Analytica Chimica Acta 513 (2004) 119–123
New potentiometric microbial biosensor for ethanol determination in alcoholic beverages Lucian Rotariu∗ , Camelia Bala, Vasile Magearu Laboratory of Quality Control and Process Monitoring, Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 90 Sos. Panduri, 050663 Bucharest 5, Romania Received 2 July 2003; accepted 16 December 2003 Available online 12 February 2004
Abstract A potentiometric oxygen electrode was successfully used to produce a microbial biosensors with an extended response range, based on immobilised yeast cells (Saccharomyces ellipsoideus). The response for ethanol takes ca. 7 min for the steady-state method and 2 min for kinetic measurements. A linear calibration achieved over more than two decades of ethanol concentration. Selective determination of ethanol in the presence of non-volatile compounds was achieved using a second PTFE membrane to cover the biocatalytic layer. © 2004 Elsevier B.V. All rights reserved. Keywords: Ethanol; Microbial biosensor; Potentiometry; Food analysis
1. Introduction Ethanol is not considered food but is very often used in the human nourishment. Inside the human body, ethanol is completely oxidised or can be partially eliminated through skin, respiration or urine. Ethanol ingestion can affect the nervous system, circulatory system or digestive system. Excessive consumption could cause coma or even death. Alcohol determination, from this point of view, are important in clinical analysis [1]. In industry, determination of ethanol is of interest in preparing alcoholic beverages, in some biotechnological processes and in the cosmetics industry. Quality control of alcoholic beverages such as beer, wine, liquor and spirits implies measurement of the alcoholic compounds’ concentration in general, and ethanol concentration in particular. Ethanol could also represent a quality indication for food when ethanol is the product of food degradation. The spectrometric and chlromatographic methods [2–5] developed for this analyte are expensive and sometimes require sample pre-treatment. The food and drink industry require rapid determination methods for routine analysis in the quality control of their products [6]. ∗ Corresponding author. Tel.: +40-21-410-4888; fax: +40-21-410-4888. E-mail address: [email protected] (L. Rotariu).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.12.048
Enzymatic methods and especially enzyme biosensors present some advantage like high selectivity and specificity, and relative low cost of construction and storage [7]. Enzymatic methods for ethanol determination use only one of two enzymes, alcohol oxidase (AOD) and alcohol dehydrogenase (ADH). spectrometric [8,9], chemiluminescence [10,11] or electrometric methods were developed for AOD. Due to the lower selectivity of AOD, this enzyme was used in realisation of liquid chromatography detectors for alcohols [12,13]. ADH has better selectivity for ethanol and has been used for enzymatic biosensor development. Spectrometric methods were also described based on colorimetric [9], fluorimetric [8] or chemiluminescence [10] NADH measurements. The main problems for an ADH-based biosensor is the large amount of coenzyme required, the low stability of the enzyme and the relatively high applied potential for amperometric determination [14–17]. Mediators [18–21] have also been used for ethanol biosensor design with promising results. Bacteria and yeasts are recognised as organisms which metabolise all kinds of substrates. Amperometric and potentiometric microbial and hybrid biosensors for sugars were previously reported [22,23] based on different types of oxygen electrode [24]. Based on micro-organism like T. brassicae, S. cerevisiae, Acetobacter aceti or Acetobacter xylinnium, different types of microbial biosensor for ethanol were developed using amperometric [25,26], potentiomet-
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ric [27] or conductometric [28] detectors. Applications for quantitation of ethanol in beer and wine were also reported [29,30]. To enhance the selectivity of microbial biosensors, several approaches were used. The possible methods for enhancement of the selectivity of microbial biosensors have been well reviewed [31,32], including induction of transport/metabolic systems, inhibition of undesired transport/metabolic pathways, the coupling of enzymes with immobilised cells, and the use of anti-interference layers [22,23]. Application of the gas-permeable membrane or cellulose acetate membrane effectively increases the selectivity of microbial biosensors [33,34]. This paper describes an amperometric microbial biosensor for determination of ethanol. Incubation with ethanol was used to improve the selectivity of the microbial biosensor. High selectivity was achieved by using a second PTFE membrane to cover the biocatalytic yeast layer.
2. Experimental 2.1. Materials A yeast strain of Saccharomyces ellipsoideus, purchased from the University of Galati (Romania), was used as the biocatalyst in the microbial sensor; all other reagents were of analytical grade. Standard solution of ethanol were prepared in disodium hydrogenphosphate–critic acid buffer, 0.5 M. 2.2. Preparation of microbial electrode and assay procedure The pre-treatment and immobilisation of yeast cells has been described elsewhere [23]. The oxygen sensor MF-2100 consists in a platinum electrode, and an Ag/AgCl electrode, as reference electrode, covered with an oxygen permeable membrane. The internal electrolyte is based on manganese hydroxide in 0.1 M KCl. Potentiometric measurements were realised using an electrochemical workstation BAS 100B/W (Bioanalytical System, West Lafayette, IN), and data display and recording were supported by BAS electrochemical software version 3.2. Spectrometric measurements were performed with a JASCO Uv-Vis 540 spectromphotometer. The immobilised micro-organisms were placed on the oxygen membrane and covered with a dialysis membrane (DM) and fixed with a rubber ring. All the determinations were carried out in a 10 ml measuring cell and all the solutions were previously saturated with oxygen. Before every determination, the biosensor was kept in oxygen-saturated phosphate buffer solution. After the output signal of the microbial sensor became stable, the sensor was removed to a buffered standard solution of ethanol (saturated with oxygen).
Ethanol assimilation by the yeast cell is accompanied by oxygen consumption in the respiratory process. Oxygen consumption caused a decrease in dissolved oxygen concentration around the cell membrane and consequently a decrease in output signal. The decrease of oxygen concentration was taken as the measure of ethanol concentration in the sample. Selective determination of ethanol in the presence of non-volatile compounds was realised by adding a second PTFE membrane.
3. Results and discussion 3.1. Optimisation of the micro-organism concentration in the biocatalytic layer The membranes with immobilised yeast were tested on a 0.5 mM ethanol solution at 25 ◦ C and pH 7. Response curves were registered as the difference between steady-state signal and base signal in oxygen-saturated buffer, as represented in Fig. 1. Low immobilised cell concentrations do not modify the baseline of the biosensor in the presence of ethanol solution. Increasing the cell concentration on the surface of the membrane leads to a very low base signal for the microbial biosensor. For a very high concentration of yeast cells, the oxygen electrode could not detect dissolved oxygen around the biocatalytic membrane. Membranes made by depositing 0.5 ml of a cell suspension with absorbance of 0.1 showed the maximum variation of the potential. These membranes were used for further experiments. 3.2. Response curve of the microbial biosensor The microbial sensor was immersed in an oxygen-saturated buffer solution (pH 7, t = 25 ◦ C) for equilibration for about 5 min. After a steady-state signal was obtained, ethanol
Fig. 1. Optimisation of yeast cell concentration (temperature: 25 ◦ C, pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
L. Rotariu et al. / Analytica Chimica Acta 513 (2004) 119–123
Fig. 2. Effect of temperature on biosensor response (pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
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Fig. 3. Biosensor calibration plot (steady state, temperature: 25 ◦ C, pH 7). Each point represents the average of five determinations.
3.5. Calibration solutions were added in order to obtain final concentrations in the range 0.05–5 mM. Decrease of the oxygen concentration around the biocatalytic membrane, due to ethanol assimilation, was registered, and the potential was stabilised in 5–7 min depending on the ethanol concentration. This response time is longer than for the amperometric biosensor [34] due to the longer response time of the potentiometric oxygen electrode [24] and due to the presence of the second gas-permeable membrane between the sample and the yeast cell layer. Kinetic measurements could be made in the first 2 min. 3.3. Effect of temperature The respiration activity of the yeast cells depends on the presence of a carbon source and on the temperature. Fig. 2 shows the effect of temperature upon the response of the microbial sensor, as the sample temperature was gradually increased. The sample was kept for 5 min at the selected temperature before measurement. A temperature of 25 ◦ C was chosen as the working temperature. A lower baseline for the oxygen electrode, poorer reproducibility and a limited response range for sucrose was observed at higher temperatures.
Response curves for ethanol concentrations between 0.02 and 50 mM were recorded and calibration graphs were produced using steady-state and kinetic methods. Fig. 3 shows the graph for the steady-state method at pH 7 and 25 ◦ C. Each point represents the average for five determinations. A linear calibration plot was obtained. The linear regression parameters (y = ax + b) are a = 139.0 ± 2.3,
b = 26.51 ± 0.71
with the correlation coefficient R = 0.9978 (n = 40). The slope of 26 mV per decade shows a sub-Nernstain response of the biosensor, a comparable sensitivity with the potentiometric biosenser for sucrose [23]. The detection limit corresponds to an ethanol concentration of 5.7 M. A calibration plot for the kinetic method also shows a linear relationship between the potential and the ethanol concentration with the following parameters: a = 62.0 ± 1.1,
b = 12.85 ± 0.32,
R = 0.9981
The sensitivity for the kinetic method is about half that of the steady-state method but there was a good correlation for both methods. 3.6. Interferences
3.4. Effect of pH The effect of pH on the response was investigated in the pH range 4–8 at 25 ◦ C. The response to standard solution of 0.5 mM ethanol was recorded and no significant differences were registered in this pH range. This is a normal behaviour taking into account that the sample is separated from the yeast cell layer by the gas-permeable membrane, which does not allow ion exchange between the external and internal parts of the membrane. All further measurements were carried out at pH 7.
The selectivity of the microbial biosensor was studied on some common organic compounds present in alcoholic beverages: methanol, propanol, iso-propanol, acetic acid and glucose. Measurements were performed with yeast cells immobilised on a DM and on a PTFE membrane (TM), at 25 ◦ C, pH 7 for concentrations of 0.5 mM for each compound. The results presented in Fig. 4 show that only glucose interferes in the determination of ethanol. All the volatile organic compounds do not interfere. A second
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Fig. 4. Selectivity of the microbial biosensor for ethanol (0.5 mM, 25 ◦ C, pH 7). Fig. 6. Comparative determination of ethanol in alcoholic beverages.
anti-interference enzymatic layer based on GOD and CAT is the usual way to eliminate the interference of glucose for almost all kind of biosensors. In this case, the substrate is a volatile compound and use of a gas-permeable membrane represents the most convenient way to eliminate the interference of glucose. Using a PTFE membrane (TM) the microbial biosensor responds selectively to ethanol in the presence of all the other compounds tested. The use of this membrane affects the response time of the biosensor. Independence from external factors such as pH and contamination represent the main advantages of separation of the biocatalytic layer from the sample solution.
In between, the sensors was kept in buffer solution at 4 ◦ C. After 5 days, the signal has decreased to 70% of its initial value in the first day, and reached 40% after 8 days. Ethanol concentration in alcoholic beverages was determined using the microbial biosensor. Dilutions between 40 and 500 times were performed, depending on the beverage. The results were compared with the enzymatic spectrophotometric method. The correlation coefficient of the experimental data, 0.9972 (Fig. 6) shows a good correlation between the results obtained by the biosensor and spectrometric methods. The coefficient of variation was >6% for the biosensor and >2.5% for the spectrophotometric method, for determinations carried out on the same day.
3.7. Stability Biosensor stability was tested on 0.5 mM ethanol, pH 7, 25 ◦ C, for 8 days with five determinations per day (Fig. 5).
Fig. 5. Biosensor stability (temperature: 25 ◦ C, pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
4. Conclusions A microbial biosensor for ethanol based on yeast cell of S. ellipsoideus and potentiometric oxygen electrode was produced and characterised. The main advantage of this biosensor is its construction simplicity and low cost. Optimum working conditions were pH 7 and temperature of 25 ◦ C. A fast response of about 2 min was registered for yeast immobilised on a DM and about 7 min for selective biosensor with yeast immobilised on a PTFE membrane. The interference of glucose in ethanol determination is significant even after yeast incubation with ethanol before biosensor preparation. Use of the PTFE membrane allows one to prepare a highly selective biosensor for ethanol. A linear calibration plot was recorded for more than three decades of ethanol concentration, being the most extended calibration plot of all microbial biosensor calibrations previously reported [34,35]. A comparable detection limit was obtained with that of a similar amperometric biosensor described elsewhere [23]. Comparable sensitivity and good correlation with the enzymatic spectrophotometric method were observed.
L. Rotariu et al. / Analytica Chimica Acta 513 (2004) 119–123
References [1] M. Cotr˘au, M. Proca, Toxicologie analitic˘a, Ed. Medical˘a, Bucure¸sti, 1988. [2] Gh. Ghimicescu, Chimia si analiza alimentelor, bauturilor si condimentelor, Ed. Junimea, Iasi, 1977. [3] S.P. Clarkson, I.H.L. Onrnod, F.R. Sharpe, J. Inst. Brew. 101 (1995) 191–193. [4] H. Liden, A.R. Vijayakumar, L. Gorton, G. Marko-Varga, J. Pharm. Biomed. Anal. 17 (1998) 1111–1128. [5] K. Johansson, G. Jönsson-Pettersson, L. Gorton, G. Marko-Varga, E. Csöregi, J. Biotechnol. 31 (1993) 301–316. [6] G. Wagner, G.G. Guilbault, Food Biosensors Analysis, Marcel Dekker, New York, 1994. [7] L.D. Mello, L.T. Kubota, Food Chem. 77 (2002) 237–256. [8] E. Forster, M. Silva, M. Otto, D. Perez-Bendito, Anal. Chim. Acta 274 (1993) 109–116. [9] S.Y. Chung, J.R. Vercellotti, T.H. Sanders, J. Agric. Food Chem. 43 (6) (1995) 1545–1548. [10] A. Nabi, P.J. Worsfold, Talanta 111 (1986) 1–7. [11] X. Xie, A.A. Subiman, G.G. Guilbault, Z. Yang, Anal. Chim. Acta 266 (1992) 325–329. [12] G. Marko-Varga, K. Johansson, L. Gorton, J. Chromatogr. A 660 (1–2) (1994) 153–167. [13] K. Lohanson, G. Jonsson-Pettersson, L. Gorton, G. Marko-Varga, E. Csoregi, J. Biotechnol. 31 (1993) 301–316. [14] S. Miertus, J. Katrlik, A. Pizzariello, M. Stredansk’y, J. Svitel, J. Svorc, Biosens. Bioelectron. 13 (7–8) (1998) 911–923. [15] L. Gorton, E. Dominguez, B. Hahn-Hagerdel, H.L. Lan, Y. Okamoto, P.D. Hale, T.A. Skutheim, Anal. Chim. Acta 249 (1993) 43. [16] J. Katrlik, J. Svorc, M. Stred’ansky, S. Miertus, Biosens. Bioelectron. 13 (2) (1998) 181–191. [17] L. Geng, L.I. Boguslavsky, I.P. Kovalev, S.K. Sahni, H. Kalash, T.A. Skotheim, H.S. Lee, V. Laurinavicius, Biosens. Bioclectron. 11 (12) (1996) 1267–1275. [18] P.C. Pandey, S. Upadhyay, B.C. Upadhyay, H.C. Pathak, Anal. Biochem. 260 (2) (1998) 195–203.
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[19] E. Lorenzo, F. Pariente, L. Hernandez, F. Tobalina, M. Darder, Q. Wu, M. Maskus, H.D. Abruna, Biosens. Bioelectron. 13 (3–4) (1998) 319–332. [20] B. Leca, J. Marty, Biosens. Bioelectron. 12 (11) (1997) 1083–1088. [21] M.J.L. Castanon, A.J.M. Ordieres, P.T. Blanco, Biosens. Bioelectron. 12 (6) (1997) 511–520. [22] L. Rotariu, C. Bala, V. Magearu, Rev. Roum. Chim. 45 (1) (2000) 21–26. [23] L. Rotariu, C. Bala, V. Magearu, Anal. Chim. Acta 458 (1) (2002) 215–222. [24] L. Rotariu, C. Bala, S. Fleschin, V. Magearu, Anal. Univ. Buc., Anul III (serie noua), 1994, 45–48. [25] M.V. Gonchar, M.M. Maidan, O.M. Moroz, J.R. Woodward, A.A. Sibirny, Biosens. Bioelectron. 13 (9) (1998) 945–952. [26] A.N. Reshetilov, A.V. Lobanov, N.O. Morozova, S.H. Gordon, R.V. Greene, T.D. Leathers, Biosens. Bloelectron. 13 (7–8) (1998) 787– 793. [27] Y.I. Korpan, M.V. Gonchar, A.P. Soldatkin, N.F. Starodub, A.K. Sandrovski, A.A. Sibirnyi, A.V. El’skaia, Ukr. Biokhim. Zh. 64 (3) (1992) 96–100. [28] Y.I. Korpan, S.V. Dzyadevich, V.P. Zharova, A.V. El’skaya, Ukr. Biokhim. Zh. 66 (1) (1994) 78–82. [29] I. Karube, in: A.P.F. Turner, I. Karube, G.S. Wilson (Eds.), Biosensors: Fundamentals and Applications, Mir Publishers, Moscow, 1992, pp. 20–33. [30] I. Karube, E. Tamiya, K. Sode, K. Yokoyama, Y. Kitagawa, H. Suzuki, Y. Asanao, Application of microbiological sensors in fermentation processes, Anal. Chim. Acta 231 (1988) 69–77. [31] J. Racek, Cell-Based Biosensors, Technomic Publishing, Lancaster, 1994. [32] K. Riedel, Biochemical fundamentals and improvement of the selectivity of microbial sensor—a mini review, Bioelectrochem. Bioenerg. 25 (1991) 19–30. [33] M. Hikuma, T. Kubo, T. Yasuda, I. Karube, S. Suzuki, Microbial electrode sensor for alcohols, Biotechnol. Bioeng. 21 (1979) 1845– 1853. [34] L. Rotariu, C. Bala, Anal. Lett. 36 (11) (2003) 2455–2467. [35] L. Rotariu, C. Bala, V. Magearu, Anal. Univ. Buc., Anul XII, I–II (2003) 69–76.
New potentiometric microbial biosensor for ethanol determination in alcoholic beverages Lucian Rotariu∗ , Camelia Bala, Vasile Magearu Laboratory of Quality Control and Process Monitoring, Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 90 Sos. Panduri, 050663 Bucharest 5, Romania Received 2 July 2003; accepted 16 December 2003 Available online 12 February 2004
Abstract A potentiometric oxygen electrode was successfully used to produce a microbial biosensors with an extended response range, based on immobilised yeast cells (Saccharomyces ellipsoideus). The response for ethanol takes ca. 7 min for the steady-state method and 2 min for kinetic measurements. A linear calibration achieved over more than two decades of ethanol concentration. Selective determination of ethanol in the presence of non-volatile compounds was achieved using a second PTFE membrane to cover the biocatalytic layer. © 2004 Elsevier B.V. All rights reserved. Keywords: Ethanol; Microbial biosensor; Potentiometry; Food analysis
1. Introduction Ethanol is not considered food but is very often used in the human nourishment. Inside the human body, ethanol is completely oxidised or can be partially eliminated through skin, respiration or urine. Ethanol ingestion can affect the nervous system, circulatory system or digestive system. Excessive consumption could cause coma or even death. Alcohol determination, from this point of view, are important in clinical analysis [1]. In industry, determination of ethanol is of interest in preparing alcoholic beverages, in some biotechnological processes and in the cosmetics industry. Quality control of alcoholic beverages such as beer, wine, liquor and spirits implies measurement of the alcoholic compounds’ concentration in general, and ethanol concentration in particular. Ethanol could also represent a quality indication for food when ethanol is the product of food degradation. The spectrometric and chlromatographic methods [2–5] developed for this analyte are expensive and sometimes require sample pre-treatment. The food and drink industry require rapid determination methods for routine analysis in the quality control of their products [6]. ∗ Corresponding author. Tel.: +40-21-410-4888; fax: +40-21-410-4888. E-mail address: [email protected] (L. Rotariu).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.12.048
Enzymatic methods and especially enzyme biosensors present some advantage like high selectivity and specificity, and relative low cost of construction and storage [7]. Enzymatic methods for ethanol determination use only one of two enzymes, alcohol oxidase (AOD) and alcohol dehydrogenase (ADH). spectrometric [8,9], chemiluminescence [10,11] or electrometric methods were developed for AOD. Due to the lower selectivity of AOD, this enzyme was used in realisation of liquid chromatography detectors for alcohols [12,13]. ADH has better selectivity for ethanol and has been used for enzymatic biosensor development. Spectrometric methods were also described based on colorimetric [9], fluorimetric [8] or chemiluminescence [10] NADH measurements. The main problems for an ADH-based biosensor is the large amount of coenzyme required, the low stability of the enzyme and the relatively high applied potential for amperometric determination [14–17]. Mediators [18–21] have also been used for ethanol biosensor design with promising results. Bacteria and yeasts are recognised as organisms which metabolise all kinds of substrates. Amperometric and potentiometric microbial and hybrid biosensors for sugars were previously reported [22,23] based on different types of oxygen electrode [24]. Based on micro-organism like T. brassicae, S. cerevisiae, Acetobacter aceti or Acetobacter xylinnium, different types of microbial biosensor for ethanol were developed using amperometric [25,26], potentiomet-
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ric [27] or conductometric [28] detectors. Applications for quantitation of ethanol in beer and wine were also reported [29,30]. To enhance the selectivity of microbial biosensors, several approaches were used. The possible methods for enhancement of the selectivity of microbial biosensors have been well reviewed [31,32], including induction of transport/metabolic systems, inhibition of undesired transport/metabolic pathways, the coupling of enzymes with immobilised cells, and the use of anti-interference layers [22,23]. Application of the gas-permeable membrane or cellulose acetate membrane effectively increases the selectivity of microbial biosensors [33,34]. This paper describes an amperometric microbial biosensor for determination of ethanol. Incubation with ethanol was used to improve the selectivity of the microbial biosensor. High selectivity was achieved by using a second PTFE membrane to cover the biocatalytic yeast layer.
2. Experimental 2.1. Materials A yeast strain of Saccharomyces ellipsoideus, purchased from the University of Galati (Romania), was used as the biocatalyst in the microbial sensor; all other reagents were of analytical grade. Standard solution of ethanol were prepared in disodium hydrogenphosphate–critic acid buffer, 0.5 M. 2.2. Preparation of microbial electrode and assay procedure The pre-treatment and immobilisation of yeast cells has been described elsewhere [23]. The oxygen sensor MF-2100 consists in a platinum electrode, and an Ag/AgCl electrode, as reference electrode, covered with an oxygen permeable membrane. The internal electrolyte is based on manganese hydroxide in 0.1 M KCl. Potentiometric measurements were realised using an electrochemical workstation BAS 100B/W (Bioanalytical System, West Lafayette, IN), and data display and recording were supported by BAS electrochemical software version 3.2. Spectrometric measurements were performed with a JASCO Uv-Vis 540 spectromphotometer. The immobilised micro-organisms were placed on the oxygen membrane and covered with a dialysis membrane (DM) and fixed with a rubber ring. All the determinations were carried out in a 10 ml measuring cell and all the solutions were previously saturated with oxygen. Before every determination, the biosensor was kept in oxygen-saturated phosphate buffer solution. After the output signal of the microbial sensor became stable, the sensor was removed to a buffered standard solution of ethanol (saturated with oxygen).
Ethanol assimilation by the yeast cell is accompanied by oxygen consumption in the respiratory process. Oxygen consumption caused a decrease in dissolved oxygen concentration around the cell membrane and consequently a decrease in output signal. The decrease of oxygen concentration was taken as the measure of ethanol concentration in the sample. Selective determination of ethanol in the presence of non-volatile compounds was realised by adding a second PTFE membrane.
3. Results and discussion 3.1. Optimisation of the micro-organism concentration in the biocatalytic layer The membranes with immobilised yeast were tested on a 0.5 mM ethanol solution at 25 ◦ C and pH 7. Response curves were registered as the difference between steady-state signal and base signal in oxygen-saturated buffer, as represented in Fig. 1. Low immobilised cell concentrations do not modify the baseline of the biosensor in the presence of ethanol solution. Increasing the cell concentration on the surface of the membrane leads to a very low base signal for the microbial biosensor. For a very high concentration of yeast cells, the oxygen electrode could not detect dissolved oxygen around the biocatalytic membrane. Membranes made by depositing 0.5 ml of a cell suspension with absorbance of 0.1 showed the maximum variation of the potential. These membranes were used for further experiments. 3.2. Response curve of the microbial biosensor The microbial sensor was immersed in an oxygen-saturated buffer solution (pH 7, t = 25 ◦ C) for equilibration for about 5 min. After a steady-state signal was obtained, ethanol
Fig. 1. Optimisation of yeast cell concentration (temperature: 25 ◦ C, pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
L. Rotariu et al. / Analytica Chimica Acta 513 (2004) 119–123
Fig. 2. Effect of temperature on biosensor response (pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
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Fig. 3. Biosensor calibration plot (steady state, temperature: 25 ◦ C, pH 7). Each point represents the average of five determinations.
3.5. Calibration solutions were added in order to obtain final concentrations in the range 0.05–5 mM. Decrease of the oxygen concentration around the biocatalytic membrane, due to ethanol assimilation, was registered, and the potential was stabilised in 5–7 min depending on the ethanol concentration. This response time is longer than for the amperometric biosensor [34] due to the longer response time of the potentiometric oxygen electrode [24] and due to the presence of the second gas-permeable membrane between the sample and the yeast cell layer. Kinetic measurements could be made in the first 2 min. 3.3. Effect of temperature The respiration activity of the yeast cells depends on the presence of a carbon source and on the temperature. Fig. 2 shows the effect of temperature upon the response of the microbial sensor, as the sample temperature was gradually increased. The sample was kept for 5 min at the selected temperature before measurement. A temperature of 25 ◦ C was chosen as the working temperature. A lower baseline for the oxygen electrode, poorer reproducibility and a limited response range for sucrose was observed at higher temperatures.
Response curves for ethanol concentrations between 0.02 and 50 mM were recorded and calibration graphs were produced using steady-state and kinetic methods. Fig. 3 shows the graph for the steady-state method at pH 7 and 25 ◦ C. Each point represents the average for five determinations. A linear calibration plot was obtained. The linear regression parameters (y = ax + b) are a = 139.0 ± 2.3,
b = 26.51 ± 0.71
with the correlation coefficient R = 0.9978 (n = 40). The slope of 26 mV per decade shows a sub-Nernstain response of the biosensor, a comparable sensitivity with the potentiometric biosenser for sucrose [23]. The detection limit corresponds to an ethanol concentration of 5.7 M. A calibration plot for the kinetic method also shows a linear relationship between the potential and the ethanol concentration with the following parameters: a = 62.0 ± 1.1,
b = 12.85 ± 0.32,
R = 0.9981
The sensitivity for the kinetic method is about half that of the steady-state method but there was a good correlation for both methods. 3.6. Interferences
3.4. Effect of pH The effect of pH on the response was investigated in the pH range 4–8 at 25 ◦ C. The response to standard solution of 0.5 mM ethanol was recorded and no significant differences were registered in this pH range. This is a normal behaviour taking into account that the sample is separated from the yeast cell layer by the gas-permeable membrane, which does not allow ion exchange between the external and internal parts of the membrane. All further measurements were carried out at pH 7.
The selectivity of the microbial biosensor was studied on some common organic compounds present in alcoholic beverages: methanol, propanol, iso-propanol, acetic acid and glucose. Measurements were performed with yeast cells immobilised on a DM and on a PTFE membrane (TM), at 25 ◦ C, pH 7 for concentrations of 0.5 mM for each compound. The results presented in Fig. 4 show that only glucose interferes in the determination of ethanol. All the volatile organic compounds do not interfere. A second
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Fig. 4. Selectivity of the microbial biosensor for ethanol (0.5 mM, 25 ◦ C, pH 7). Fig. 6. Comparative determination of ethanol in alcoholic beverages.
anti-interference enzymatic layer based on GOD and CAT is the usual way to eliminate the interference of glucose for almost all kind of biosensors. In this case, the substrate is a volatile compound and use of a gas-permeable membrane represents the most convenient way to eliminate the interference of glucose. Using a PTFE membrane (TM) the microbial biosensor responds selectively to ethanol in the presence of all the other compounds tested. The use of this membrane affects the response time of the biosensor. Independence from external factors such as pH and contamination represent the main advantages of separation of the biocatalytic layer from the sample solution.
In between, the sensors was kept in buffer solution at 4 ◦ C. After 5 days, the signal has decreased to 70% of its initial value in the first day, and reached 40% after 8 days. Ethanol concentration in alcoholic beverages was determined using the microbial biosensor. Dilutions between 40 and 500 times were performed, depending on the beverage. The results were compared with the enzymatic spectrophotometric method. The correlation coefficient of the experimental data, 0.9972 (Fig. 6) shows a good correlation between the results obtained by the biosensor and spectrometric methods. The coefficient of variation was >6% for the biosensor and >2.5% for the spectrophotometric method, for determinations carried out on the same day.
3.7. Stability Biosensor stability was tested on 0.5 mM ethanol, pH 7, 25 ◦ C, for 8 days with five determinations per day (Fig. 5).
Fig. 5. Biosensor stability (temperature: 25 ◦ C, pH 7, ethanol: 0.5 mM). Each point represents the average of three determinations.
4. Conclusions A microbial biosensor for ethanol based on yeast cell of S. ellipsoideus and potentiometric oxygen electrode was produced and characterised. The main advantage of this biosensor is its construction simplicity and low cost. Optimum working conditions were pH 7 and temperature of 25 ◦ C. A fast response of about 2 min was registered for yeast immobilised on a DM and about 7 min for selective biosensor with yeast immobilised on a PTFE membrane. The interference of glucose in ethanol determination is significant even after yeast incubation with ethanol before biosensor preparation. Use of the PTFE membrane allows one to prepare a highly selective biosensor for ethanol. A linear calibration plot was recorded for more than three decades of ethanol concentration, being the most extended calibration plot of all microbial biosensor calibrations previously reported [34,35]. A comparable detection limit was obtained with that of a similar amperometric biosensor described elsewhere [23]. Comparable sensitivity and good correlation with the enzymatic spectrophotometric method were observed.
L. Rotariu et al. / Analytica Chimica Acta 513 (2004) 119–123
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