Development of Electrochemical Microbial Biosensor for Ethanol Based on Aspergillus niger Sreenath Subrahmanyam,* +,++++ Kumaran Shanmugam, + T.V. Subramanian, + M. Murugesan, ++ V. Murali Madhav, +++ and D. Jeyakumar ++ + Department of Chemical Engineering, A.C. College of Technology, Anna University, Madras - 600 025, Tamil Nadu State, India ++ Electronics and Electrocatalysis Division, Central Electrochemical Research Institute, Karaikudi - 630 006, Tamil Nadu State, India +++ Division of BioSensors, Central Food Technological Research Institute, Mysore, Karnataka State, India ++++ Present address: Cranfield Biotechnology Centre, Cranfield University at Silsoe, MK45 4DT, U.K.; e-mail: sri@cranfield.ac.uk Received: July 26, 2000 Final version: November 9, 2000 Abstract Electrochemical biosensors have become very important tools in analytical chemistry because of their advantages like accuracy, great sensitivity and easy handling. This article reports on the electrochemical detection of ethanol based on the fungus Aspergillus niger . The fungus was isolated from polyurethane waste, at a factory in India. The fungal assimilation of ethanol came to light, when detailed bio- chemical characterization of the fungus for alcohols, organic acids, and amino acids was studied using cyclic voltammetry. The fungus was cultured in Czepek-Dox media. A dissolved oxygen (DO) probe was fabricated using a gold cathode, a platinum counter electrode and Ag=AgCl reference electrode. The biosensor employed for the assimilation characterization was fabricated by coupling the immobilized membrane with the DO probe using dialysis tubing. A microbial dispersion containing 0.2 g (wet weight) of the organisms=mL was used for immobilization upon cellulose nitrate membrane. The membrane retaining the fungus was placed on the Teflon membrane of the oxygen electrode so that the fungus was trapped between the two membranes. The maximum detectable concentration was 35.3 ppm (v=v) and the linear range was between 1 and 32 ppm (v=v) of ethyl alcohol. By dipping the assembly with the microbe in phosphate buffer, the sensor was found to have an extended lifetime of 90 days with about 1000 determinations. Keywords: Ethanol, Biosensor, Aspergillus niger, Isolation, Biochemical characterization 1. Introduction A detailed investigation on the biochemical characteristics of the microorganisms in question is necessary in order to develop a biosensor for enhanced stability and selectivity. Research on biosensors has grown phenomenally in recent years. Many microorganisms have been characterized for their potential use in biosensors by testing their responses to many different substrates and substrate mixtures. The quantitative nature and low analyte response of the microorganisms have been studied quite in detail. Microbial sensors for monitoring various pollutants have been developed [1], where the microorganisms are in direct contact with a transducer, converting the biochemical signal into an electrical response. Characterizations for the microorganisms have been studied based on the assimilation of substrates, recording the respiratory activity of the microorganisms [2–6]. Microbial sensors comprised of immobilized whole cells and an oxygen probe have been used for determination of assimilable sugars [7], acetic acid [8], alcohol [9] vitamins, antibiotics, peptides, enzyme activities and biochemical oxygen demand [10–18], cofactors, inhibitors and for measurement of enzyme activities [19–21], and for determination of glutamic acid based on the decarboxylation of glutamic acid by glutamate decar- boxylase by measuring the carbon dioxide produced by a carbon dioxide gas sensing probe [22]. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actino- mycetes and activated sludges have been tested with various substrates. Key metabolites, or rate of their overall metabolic reactions ATP, NADH, cytochrome content and DNA [23–29], are suitable for describing the physiological state. There have been reports on simultaneous measurement of two parameters, the content of proteins and nucleic acids and the determination of complex variables like biodegradable compounds and muta- genic substances in waste water [30–34]. It also has been found that a very low microbial loading of the biosensor is a pre- requisite for a kinetically controlled respiration electrode [35] and such sensors coupled with suitable immobilization of the microorganisms as well as thin membranes have to be used [36]. The sensitivity of this type of sensor is mainly determined by the cell activity and the advantage is that one could attain response times of about 15 s by very low ‘microbe loading’ of the sensor [37]. A combination of an electrode system with very low amounts of whole cells, resulting in a decrease of diffusion resistance that leads to very fast detection of changes in the respiration rates (1–5 s), in contrast to microbial electrodes for assimilation tests, with response times from 5 to 30 min has been reported previously [37, 38]. This research article discusses the possible applications of the fungus Aspergillus niger, which has been isolated from one of the factories manufacturing polyurethane in Vellore, Tamilnadu State, India. A complete biochemical characterization was done for this fungus, (data not shown). The fungus showed very high assimilation for the alcohol, and more importantly, the fungus showed highest response for ethanol and hence was further probed for specific assimilation of the substrate ethanol. The novel aspects of this work are that the fungus showed good response for very high concentrations of ethanol, higher than the ones reported before for microbes, and the stability of the microbes giving us an extended lifetime for the sensor. 2. Experimental 2.1. Materials Potassium dihydrogen orthophosphate was purchased from Ranbaxy Laboratories, Bombay, India, and all the other chemi- cals were purchased from Loba Chemie, Bombay, India. All the analyses were done using millipore water. 944 Electroanalysis 2001, 13, No. 11 # WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 1040-0397/01/1107–0944 $17.50þ.50=0