Ultrasonic analysis of kinetic mechanism of hydrolysis of cellobiose by ß-glucosidase.

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a ß-glucosidase from Aspergillus niger (Novozyme 188) at 50°C and pH 4.9. This technique is noninvasive, it does not require optical transparency and is suitable to continuously monitor the time dependence of the reaction progress in a broad range of experimental conditions. The time profiles of the amount of glucose released and the reaction rate were obtained from the time profile of ultrasonic velocity. The results are in good agreement with a discontinuous glucose assay (hexokinase method). The kinetic parameters of the reaction were estimated by fitting the ultrasonic time profiles of the reaction rates to several inhibition models. In addition, the equilibrium constant for the reaction of hydrolysis of cellobiose and the molar Gibbs free energy of hydrolysis were determined from the ultrasonic time profiles of concentration of glucose in the reverse reaction (glucose condensation). The results suggest the existence of more complex mechanisms regulating the activity of cellobiase than the combination of simple inhibitions. An extended kinetic model based on two sites for the competitive inhibitor (glucose) is proposed.

Monitoring Escherichia coli growth in M63 media by ultrasonic noninvasive methods and correlation with spectrophotometric and HPLC techniques.

A low-intensity ultrasonic technique (that is noninvasive, nondestructive, and online) has been developed to monitor the growth of Escherichia coli in glucose minimal media under aerobic and anaerobic conditions. Ultrasonic time of flight (TOF) variations were correlated with microorganism growth and the disappearance of nutrients and their subsequent conversion into different metabolites. Spectrophotometric growth data and high-performance liquid chromatography (HPLC) analysis of released and consumed metabolites were compared with the ultrasonic data demonstrating that the ultrasound device presented can provide supplementary real-time noninvasive information about the metabolic processes taking place in the culture. A semiempirical model under aerobic conditions was developed to explain the TOF variations as a function of the glucose concentration and was modified to take into account the growth inhibition due to the initial glucose concentration. The inhibition effect was obtained by fitting HPLC measurements to the logistic function of Boltzmann. Under aerobic and anaerobic culture conditions, the metabolic processes were correlated with TOF experimental variations through a theoretical approach based on ultrasonic propagation in ternary mixtures.

Ultrasonic velocity assay of extracellular invertase in living yeasts.

The use of a low-intensity ultrasonic technique (noninvasive, nondestructive, on-line, and able to assess opaque samples) to monitor the kinetics of invertase hydrolysis is presented. Adiabatic compressibility has been shown to be sensitive to sugar species: ultrasonic velocity increasing as saccharose is transformed into glucose and fructose. The influence of initial sucrose mass concentration (2-60%), temperature (25-55 degrees C), pH (3.5-6.5), and number of microorganisms (10(5)-10(9) yeasts/ml) on the reaction rate, catalyzed by the extracellular invertases of intact Saccharomyces cerevisiae cells, has been measured. The results were proven to be in strict agreement with the optimal kinetic parameters of the enzyme. Ultrasonic velocity variations are explained in terms of changes of the solute concentrations in the mixture water-saccharose-glucose/fructose and calculated from the velocity of ultrasound in the corresponding pure sugar solutions. A linear relationship between the initial rate of ultrasonic velocity and the number of yeasts (enzymes) is pointed out.

On-line ultrasonic velocity monitoring of alcoholic fermentation kinetics.

In this work, fundamental aspects on the ultrasonic velocity monitoring of alcoholic fermentations in synthetic broths (glucose, fructose and sucrose) and natural media (must and wort) are reported. Results are explained in terms of monosaccharide catabolism, polysaccharide hydrolysis, gas production and microorganism growth. The effect of each one of these subprocesses upon ultrasonic velocity has been independently studied. It is shown that, regarding the sound propagation, the simplest systems behave as ternary dissolutions of sugar and ethanol in water, where, in the course of time, substrates are transformed into metabolites according to the fermentation reaction. A semi-empirical approach, based on the excess volume concept and the density and velocity measurements of binary mixtures, has been used to calculate these magnitudes in the ternary mixtures and to obtain the concentrations of the main solutes throughout the fermentations, reaching a good correlation (especially for the media of simplest composition). In all the processes analyzed, the data obtained from the ultrasonic measurements followed the changes caused by the yeast metabolism, asserting the potential of mechanical waves to monitor fermentations and, in general, biotechnological processes.

Theoretical analysis and modeling of Thickness-Expansion Mode (TEM) sensors for fluid characterization.

In this paper, the principles of Thickness-Expansion Mode (TEM) resonators for the characterization of fluids are described. From the measurement of the resonance parameters of a TEM piezoelectric transducer, the compressional acoustic impedance of gases and liquids can be determined. Since the propagation of mechanical waves into the fluid is not necessary, information in a wide range of frequencies can be obtained. Alternatively, these sensors can be driven in combination with other ultrasonic techniques to simultaneously determine the density, speed of sound and viscosity of samples. Some potential applications include the probe monitoring of processes and the characterization of fluids under harsh conditions. The main experimental criteria for the design and construction of high-resolution impedance meters (such as piezoelectric material, protective coating or thermal response) have been studied using equivalent electrical circuit modeling and finite element analysis.