Carbon Dioxide Electrode Based Yeast Biosensor: A Generic Tool to Study Microbial Metabolism

EDUARDO CORTON; A HILDING OHLSSON; M MARTÍNEZ; A. A. VIALE

Autónoma de Buenos Aires

Congreso; XVII Latinoamerican, and Argentinian Congress of Microbiology; 2004

Sociedad Argentina de Microbiologia

Biosensors are made by coupling of a biological material with an adequate transducer, electrochemical, thermal, optical, etc., to enable rapid and accurate detection of various substances. The most developed class of biosensors until now has been that of enzyme-based ones, which have found an important field of application in clinical chemistry; a good example is the determination of glucose in blood. Cell-based biosensors have been proposed for the determination of glutamic acid, nicotinic acid, simple carbohydrates, as well as other substances. Two strains of Bakers’ yeast (Saccharomyces cerevisiae) were tested in this work with similar results. Cellulose nitrate membranes (0.45 µm) were used to immobilize 5 mg of yeast over the carbon dioxide electrode membrane, and tightly fixed. Before the immobilization, the biosensor was immersed in plain buffer, until reach a constant CO2 concentration reading. Under conditions of nutrient limitation, the respiratory activity is low, necessary to guarantee the survival of the cell, and the rate of CO2 production measure define a base line. When a suitable carbon source is added, some of it is taken up by the cells and degraded. The respiration rate increases resulting in an increase in the carbon dioxide on the sensitive electrode surface, until a new steady-state is reached. The affinity of hexose transporters could be estimated considering that the transport across the membrane is the limiting step in the metabolic process. In this way, the production of CO2 must be directly related with the hexose intake. The increase of the production of CO2 by Baker’ yeast following a increase in the concentration of metabolizable carbohydrates follow the Mikaelis-Menten. D-Glucose Transport: Three types of transporters are described, low-affinity transporters (Km = 50-100 mM), moderately low affinity (Km about 10 mM), and high-affinity transporters (Km = 1-2 mM). The data obtained for the apparent Mikaelis-Menten constant (Km(app)) 6.7 mM seems to fit with the moderately low affinity glucose transporters. It must be noted that the real biological process involve activation and depression of different transporters when the extracellular glucose concentration change, therefore is more realistic to assume that the obtained kinetics value are indicative (because of it, we calculate apparent Km values), and represents and average of all the involved glucose Baker’s yeast transporters. We have demonstrated in this report that the microbial-based biosensor could be successfully applied to study and characterize kinetically the transport of saccharides across the cellular membrane, by applying the Mikaelis-Menten model.