Suspension culture in a T-flask with acoustic flow induced by ultrasonic irradiation

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.     

New insights into the mechanisms of ultrasonic emulsification in the oil–water system and the role of gas bubbles

Ultrasonic emulsification (USE) assisted by cavitation is an effective method to produce emulsion droplets. However, the role of gas bubbles in the USE process still remains unclear. Hence, in the present paper, high-speed camera observations of bubble evolution and emulsion droplets formation in oil and water were used to capture in real-time the emulsification process, while experiments with different gas concentrations were carried out to investigate the effect of gas bubbles on droplet size. The results show that at the interface of oil and water, gas bubbles with a radius larger than the resonance radius collapse and sink into the water phase, inducing (oil–water) blended liquid jets across bubbles to generate oil-in-water-in-oil (O/W/O) and water-in-oil (W/O) droplets in the oil phase and oil-in-water (O/W) droplets in the water phase, respectively. Gas bubbles with a radius smaller than the resonance radius at the interface always move towards the oil phase, accompanied with the generation of water droplets in the oil phase. In the oil phase, gas bubbles, which can attract bubbles nearby the interface, migrate to the interface of oil and water due to acoustic streaming, and generate numerous droplets. As for the gas bubbles in the water phase, those can break neighboring droplets into numerous finer ones during bubble oscillation. With the increase in gas content, more bubbles undergo chaotic oscillation, leading to smaller and more stable emulsion droplets, which explains the beneficial role of gas bubbles in USE. Violently oscillating microbubbles are, therefore, found to be the governing cavitation regime for emulsification process. These results provide new insights to the mechanisms of gas bubbles in oil–water emulsions, which may be useful towards the optimization of USE process in industry.     

Drying characteristics and quality attributes of apple slices dried by a non-thermal ultrasonic contact drying method

Drying is one of the most prevalent methods to reduce water activity and preserve foods. However, it is also the most energy-intensive food processing unit operation. Although a number of drying methods have been proposed and tested for the purpose of achieving a time- and energy-efficient drying process, almost all current drying methods still rely on thermal energy to remove moisture from the product. In this study, a novel use of power ultrasound was explored for drying of apple slices without the application of heat. The non-thermal ultrasound contact drying (US-CD) was performed in the presence of an air stream (26–40 °C) flowing over product surface to remove mist or vapor produced by the ultrasound treatment. The effects of the non-thermal US-CD, hot-air drying (HAD), and freeze drying (FD) on the changes in rehydration ratio, pH, titratable acidity, water activity, color, glass transition temperature, texture, antioxidant capacity, total phenols, and microstructures of the samples were evaluated. The moisture content of the apple slices reached below 5% (w.b.) after 75–80 min of US-CD, which was about 45% less than that of the HAD method. The antioxidant capacity and total phenol contents of the US-CD samples were significantly higher than that of the AD samples. The non-thermal ultrasonic contact drying is a promising method which has the potential to significantly reduce drying time and improve product quality.     

Ultrasound-assisted of alkali chloride separation using bulk ionic liquid membrane

An ultrasonic-assisted separation of alkali chloride (LiCl, NaCl, and KCl) salts have been carried out using of an hydrophobic ionic liquid membrane (ILM). The ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and tributyl phosphate mixture have been used as ILM. An ultrasonic probe with different frequencies (25, 100, and 250) kHz have been applied as source of ultrasound generator with different times of sonication (2, 5, and 10) min in three phases system containing feed, ILM, and receiver in osmotic U-shaped tube. Also, 250, 500, and 1000 ppm of the feed (alkali chloride) concentration have been used to separate. The frequency of 250 kHz with higher sonication time provides optimum condition for separation of LiCl with lower feed concentration. The thermodynamic properties such as density and speed of sound and the related thermodynamic properties have been calculated to optimize ILM composition (x_IL = 0.45) for ultrasound-separation.     

Ultrasound-assisted production and optimization of mini-emulsions in a microfluidic chip in continuous-flow

The use of ultrasound to generate mini-emulsions (50 nm to 1 μm in diameter) and nanoemulsions (mean droplet diameter < 200 nm) is of great relevance in drug delivery, particle synthesis and cosmetic and food industries. Therefore, it is desirable to develop new strategies to obtain new formulations faster and with less reagent consumption. Here, we present a polydimethylsiloxane (PDMS)-based microfluidic device that generates oil-in-water or water-in-oil mini-emulsions in continuous flow employing ultrasound as the driving force. A Langevin piezoelectric attached to the same glass slide as the microdevice provides enough power to create mini-emulsions in a single cycle and without reagents pre-homogenization. By introducing independently four different fluids into the microfluidic platform, it is possible to gradually modify the composition of oil, water and two different surfactants, to determine the most favorable formulation for minimizing droplet diameter and polydispersity, employing less than 500 µL of reagents. It was found that cavitation bubbles are the most important mechanism underlying emulsions formation in the microchannels and that degassing of the aqueous phase before its introduction to the device can be an important factor for reduction of droplet polydispersity. This idea is demonstrated by synthetizing solid polymeric particles with a narrow size distribution starting from a mini-emulsion produced by the device.     

Complexation of maltodextrin-based inulin and green tea polyphenols via different ultrasonic pretreatment

Ultrasound has been applied in food processing for various purpose, showing potential to advance the physical and chemical modification of natural compounds. In order to explore the effect of ultrasonic pretreatment on the complexation of inulin and tea polyphenols (TPP), different frequencies (25, 40, 80 kHz) and output power (40, 80, 120 W) were carried out. According to the comparison in particle size distribution and phenolic content of different inulin-TPP complexes, it was indicated that high-intensity ultrasonic (HIU) treatment (25 kHz, 40 W, 10 min) could accelerate the interaction of polysaccharides and polyphenols. Moreover, a series of spectral analysis including UV–Vis, FT-IR and NMR jointly evidenced the formation of hydrogen bond between saccharides and phenols. However, the primary structure of inulin and the polysaccharide skeleton were not altered by the combination. Referring to field emission scanning electron microscopy (FESEM), the morphology of ultrasound treated-complex presented a slight agglomeration in the form of bent sheets, compared to non-treated sample. The inulin-TPP complex also revealed better stability based on thermogravimetric analysis (TGA). Thus, it can be speculated from the identifications that proper ultrasonic treatment is promising to promote the complexation of some food components during processing.     

Ultrasound-assisted soil washing processes for the remediation of heavy metals contaminated soils: The mechanism of the ultrasonic desorption

Ultrasound-assisted soil washing processes were investigated for the removal of heavy metals (Cu, Pb, and Zn) in real contaminated soils using HCl and EDTA. The ultrasound-assisted soil washing (US/Mixing) process was compared with the conventional soil washing (Mixing) process based on the mechanical mixing. High removal efficiency (44.8% for HCl and 43.2% for EDTA) for the metals was obtained for the most extreme conditions (HCl 1.0 M or EDTA 0.1 M and L:S = 10:1) in the Mixing process. With the aide of ultrasound, higher removal efficiency (57.9% for HCl and 50.0% for EDTA) was obtained in the same extreme conditions and similar or higher removal efficiency (e.g., 54.7% for HCl 0.5 M and L:S = 10:1 and 50.5% for EDTA 0.05 M and L:S = 5:1) was achieved even in less extreme conditions (lower HCl or EDTA concentration and L:S ratio). Therefore, it was revealed that the US/Mixing was advantageous over the conventional Mixing processes in terms of metal removal efficiency, consumption of chemicals, amount of generated washing leachate, and volume/size of washing reactor. In addition, the heavy metals removal was enhanced for the smaller soil particles in the US/Mixing process. It was due to more violent movement of smaller particles in slurry phase and more violent sonophysical effects. In order to understand the mechanism of ultrasonic desorption, the desorption test was conducted using the paint-coated beads with three sizes (1, 2, and 4 mm) for the free and attached conditions. It was found that no significant desorption/removal of paint from the beads was observed without the movement of beads in the water including floatation, collision, and scrubbing. Thus, it was suggested that the simultaneous application of the ultrasound and mechanical mixing could enhance the physical movement of the particles significantly and the very high removal/desorption could be attained.     

Particle separation in microfluidics using different modal ultrasonic standing waves

Microfluidic technology has great advantages in the precise manipulation of micro and nano particles, and the separation of micro and nano particles based on ultrasonic standing waves has attracted much attention for its high efficiency and simplicity of structure. This paper proposes a device that uses three modes of ultrasonic standing waves to continuously separate particles with positive acoustic contrast factor in microfluidics. Three modes of acoustic standing waves are used simultaneously in different parts of the microchannel. According to the different acoustic radiation force received by the particles, the particles are finally separated to the pressure node lines on both sides and the center of the microchannel. In this separation method, initial hydrodynamic focusing and satisfying various equilibrium constraints during the separation process are the key. Through numerical simulation, the resonance frequency of the interdigital transducer, the distribution of sound pressure in the liquid, and the relationship between the interdigital electrode voltage and the output sound pressure are obtained. Finally, the entire separation process in the microchannel was simulated, and the separation of the two particles was successfully achieved. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.     

Wetting of aluminium and carbon interface during preparation of Al-Ti-C grain refiner under ultrasonic field

In the preparation of an Al-Ti-C grain refiner under an ultrasonic field, the mechanism of the wetting behaviour between Al and C was systematically investigated. The results demonstrated that the wetting behaviour was mainly dependent on the wetting of the Al melt on graphite under the ultrasonic field (physical wetting) and the formation and mass transfer of TiC (reactive wetting). The diffusion of Ti atoms and their adsorption around the graphite could contribute to the wetting of Al-C. TiC particles were formed under the high temperature caused by the cavitation effect, and they detached from the interface due to the sound pressure, which resulted in consistently sufficient contact on the wetting interface. Moreover, the wetting and spreading behaviour of the Al melt on graphite under an ultrasonic field were numerically simulated, strongly manifesting that the ultrasonic field could facilitate the wetting of the Al-C interface.     

Ultrasound-assisted solution crystallization of fotagliptin benzoate: Process intensification and crystal product optimization

The ultrasound-assisted crystallization process has promising potentials for improving process efficiency and modifying crystalline product properties. In this work, the crystallization process of fotagliptin benzoate methanol solvate (FBMS) was investigated to improve powder properties and downstream desolvation/drying performance. The direct cooling/antisolvent crystallization process was conducted and then optimized with the assistance of ultrasonic irradiation and seeding strategy. Direct cooling/antisolvent crystallization and seeding crystallization processes resulted in needle-like crystals which are undesirable for downstream processing. In contrast, the ultrasound-assisted crystallization process produced rod-like crystals and reduced the crystal size to facilitate the desolvation of FBMS. The metastable zone width (MSZW), induction time, crystal size, morphology, and process yield were studied comprehensively. The results showed that both the seeding and ultrasound-assisted crystallization process (without seeds) can improve the process yield and the ultrasound could effectively reduce the crystal size, narrow the MSZW, and shorten the induction time. Through comparing the drying dynamics of the FBMS, the small rod-shaped crystals with a mean size of 9.6 μm produced by ultrasonic irradiation can be completely desolvated within 20 h, while the desolvation time of long needle crystals with an average size of about 157 μm obtained by direct cooling/antisolvent crystallization and seeding crystallization processes is more than 80 h. Thus the crystal size and morphology were found to be the key factors affecting the desolvation kinetics and the smaller size produced by using ultrasound can benefit the intensification of the drying process. Overall, the ultrasound-assisted crystallization showed a full improvement including crystal properties and process efficiency during the preparation of fotagliptin benzoate desolvated crystals.