Abstract
A new biochemical oxygen demand (BOD) sensing method employing a double-mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione and the eukaryote Saccharomyces cerevisiae has been developed. In this study, a stirred micro-batch-type microbial sensor with a 560 μL volume and a two-electrode system was used. The chronamperometric response of this sensor had a linear response between 1 μM and 10 mM hexacyanoferrate(II) ( r 2 = 0.9995, 14 points, n = 3, average of relative standard deviation and R.S.D. av = 1.3%). Next, the optimum conditions for BOD estimation by the DM system (BOD DM) were investigated and the findings revealed that the concentration of ethanol, used to dissolve menadione, influenced the sensor response and a relationship between the sensor output and glucose glutamic acid concentration was obtained over a range of 6.6–220 mg O 2 L −1 (five points, n = 3, R.S.D. av 6.6%) when using a reaction mixture incubated for 15 min. Subsequently, the characterization of this sensor was studied. The sensor responses to 14 pure organic substances were compared with the conventional BOD 5 method and other biosensor methods. Similar results with the BOD biosensor system using Trichosporon cutaneum were obtained. In addition, the influence of chloride ion, artificial seawater and heavy metal ions on the sensor response was investigated. A slight influence of 20.0 g L −1 chloride ion and artificial seawater (18.4 g L −1 Cl −) was observed. Thus, the possibility of BOD determination for seawater was suggested in this study. In addition, no influence of the heavy metal ions (1.0 mg L −1 Fe 3+, Cu 2+, Mn 2+, Cr 3+ and Zn 2+) was observed. Real sample measurements using both river water and seawater were performed and compared with those obtained from the BOD 5 method. Finally, stable responses were obtained for 14 days when the yeast suspension was stored at 4 °C (response reduction, 93%; R.S.D. for 6 testing days, 9.1%).
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