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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.

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

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a β-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.

Ultrasonic measurement device for the characterization of microbiological and biochemical processes in liquid media

A measuring device for the characterization of liquid media based on the propagation of ultrasonic waves is presented. It is a four-channel system especially designed for monitoring microbiological and biochemical processes. The liquid samples are placed in commercial glass bottles which can be sterilized. The bottles have inlet and outlet tubes, which can be used for adding substances or extracting samples during the measuring process without interruption. Magnetic stirring can be used to keep the liquid agitated for homogenization purposes. Thermal control elements assure the temperature stability during the measurement. The liquid characterization is based on the detection of amplitude and time-of-flight changes in the sample under study. The main features, operation and performance of this ultrasonic device are analysed in this work, and some measurements and preliminary results are shown.

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.

Sound attenuation in magnetorheological fluids

In this work, the attenuation of ultrasonic elastic waves propagating through magnetorheological (MR) fluids is analysed as a function of the particle volume fraction and the magnetic field intensity. Non-commercial MR fluids made with iron ferromagnetic particles and two different solvents (an olive oil based solution and an Araldite-epoxy) were used. Particle volume fractions of up to 0.25 were analysed. It is shown that the attenuation of sound depends strongly on the solvent used and the volume fraction. The influence of a magnetic field up to 212 mT was studied and it was found that the sound attenuation increases with the magnetic intensity until saturation is reached. A hysteretic effect is evident once the magnetic field is removed.

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.

X-cut quartz crystal impedance meter for liquid characterization

A new procedure to determine the compressional acoustic impedance of liquids within ±0.05% resolution and ±0.25% accuracy is presented. This method is based on the measurement of the bandwidth of a piezoelectric transducer, vibrating in thickness-expansion mode and having a high quality factor (e.g., air-backed X-cut quartz crystal), in direct contact with the sample. In combination with other ultrasonic techniques (pulse-echo or through-transmission), it is possible to simultaneously obtain the main ultrasonic parameters (characteristic acoustic impedance, speed of sound and attenuation coefficient), and deduce important physicochemical properties of liquids, such as density and adiabatic compressibility. Experimental results for selected liquids and solutions are shown.

Ultrasonic velocity measurements in the ternary mixtures water-lactose-lactate, for the purpose of monitoring the lactic acid fermentation of lactose

A preliminary study of the lactic acid fermentation of lactose using an ultrasonic velocity technique is presented. During this fermentation, lactose (milk sugar) is transformed into lactate (lactic acid) by the action of bacteria. It is shown that the changes occurring during the course of the process can be monitored on-line by measuring the changes experienced by ultrasonic waves propagating through the fermenting media. Measurements of density and ultrasonic velocity in the ternary mixtures water-lactose-lactate and during the fermentation of a lactose solution were carried out. The ultrasonic propagation velocity has been correlated with the mass concentration of the mixture components using a semi-empirical model. These relations may be used to achieve an on-line concentration control for microbial lactic acid production.

Simultaneous observation of milk coagulation by echography, ultrasonic propagation and thermography

A cell has been developed to perform the simultaneous observation of milk coagulation by echography, local thermography and ultrasonic propagation. The chamber has five 4 MHz transducers and four thermocouples distributed along the chamber. At the upper part, an acoustic window permits the coupling of a 2.5 MHz phase array transducer linked to an echograph. The entire chamber is thermostatized at 35.00/spl deg/C into a water tank. Automatic recording of all the temperature and acoustic channels is made with typical lab instrumentation. The images are recorded in videotape to be post-processed. The results obtained are potentially useful for the dairy industry and also to understand the physicochemical process occurring during the milk gelation.

Continuous monitoring of bacterial biofilm growth using uncoated Thickness-Shear Mode resonators

Quartz Crystal Microbalances (QCM) were used to nondestructively monitor in real time the microbial growth of the bacteria Staphylococcus epidermidis (S. epidermidis) in a liquid broth. QCM, sometimes referred to as Thickness-Shear Mode (TSM) resonators, are highly sensitive sensors not only able to measure very small mass, but also non-gravimetric contributions of viscoelastic media. These devices can be used as biosensors for bacterial detection and are employed in many applications including their use in the food industry, water and environment monitoring, pharmaceutical sciences and clinical diagnosis. In this work, three strains of S. epidermidis (which differ in the ability to produce biofilm) have been continuously monitored using an array of piezoelectric TSM resonators, at 37 °C in a selective culturing media. Microbial growth was followed by measuring the changes in the crystal resonant frequency and bandwidth at several harmonics. It was shown that microbial growth can be monitored in real time using multichannel and multiparametric QCM sensors.