ArticleLiterature Review

Role of ultrasound in assisted fermentation technologies for process enhancements

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Abstract

Biological molecules are widely produced by fermentation technology using bacteria, fungi or yeast. Fermentation is a biochemical process wherein the rate of bioconversion is governed by the organisms involved. The growth of the organism is mainly limited by mass transfer rates of nutrients and gases that directly affect the product formation in fermentation. Attempts have been made to enhance the growth rate and yield using mutational, recombinant strain development approach at microbial level as well as fed batch and continuous processing approach at bioprocess level in the past. The growth rate of microbes can be accelerated by increased mass transfer rates and cell wall permeability with the use of controlled low frequency ultrasound irradiation. The present review provides insights into the application of acoustic cavitation in process intensification of fermentation approaches and the role of various factors involved are highlighted with typical examples.

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... High frequency ultrasonication generates smaller bubbles with low energy as there is less time for the bubble to grow when compared to ultrasonication with lower frequency (Leong et al., 2011). Larger the bubble size, more is the intensity generated on its implosion (Pawar and Rathod, 2020). Hence, the higher intensity achieved at a frequency of 25 kHz was more effective in weakening the adhesion of the gummy layer of the decorticated fiber. ...
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In this study, we designed, developed, and optimized an enzymatic fiber degum-ming process. First, a Partially Purified Enzyme Solution (PPES) containing pectinase, cellulase and xylanase was co-produced via ultrasound-assisted fermentation of orange peels using Bacillus subtilis ABDR01. Then, degumming with PPES was optimized for pH, temperature, incubation time and PPES concentration. Finally, three sets of ultrasound-assisted degumming experiments-chemical, enzymatic, and coupled enzymatic-chemical degumming with reduced alkali concentration (0.5% NaOH, 30 min) were performed. Chemical degumming for 2 h with 1% NaOH at 98 • C resulted in residual gum content of 5.9%, whereas ultrasound-assisted (30 min, 25 kHz) chemical degumming reduced the gum content to 3.1%. At a pH of 7.5, temperature of 45 • C, 30 h incubation and 2% PPES, gum removal efficiency of >60% and fibers with <10% residual gum were produced. Coupling the ultrasound-assisted enzymatic degumming with reduced alkali chemical degumming resulted in gum content of <3%.
... Phosphatase activity in the pre-sonicated yogurt was significantly higher than that in the unsonicated group (0.31 U g −1 ); in particular, in the yogurt pre-treated with ultrasound for 30 min, the phosphatase activity was 74.2% higher than that in the unsonicated yogurt (Supporting Information Fig. S1). Therefore, the enhancement of pesticide degradation may be attributable to the following effects of ultrasound on L. plantarum: (1) the micro-turbulence generated by ultrasound disrupted cell clusters in the inoculum, enhancing nutrient and pesticide uptake and excretion of toxic substances produced by the degradation process; 30 (2) the cavitation effect of ultrasound increased the permeability of the microbial cell membrane, enhancing nutrient transport and promoting the growth and propagation of L. plantarum; 31 and (3) ultrasound promoted the activity of phosphatase in milk, which was the main mechanism for L. plantarum to degrade organophosphorus pesticides. 32 Effect of ultrasound on fermentation efficiency Changes in lactose concentration during fermentation are shown in Fig. 4(A). ...
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Background: Ultrasound has a potential to increase microbial metabolic activity, so this study explored the stimulatory effect of ultrasound pre-treatment on the degradation of four common pesticides (fenitrothion, chlorpyrifos, profenofos, and dimethoate) during milk fermentation by Lactobacillus plantarum and its effect on yogurt quality. Results: Proper ultrasound pretreatment significantly enhanced the growth of L. plantarum. The degradation percentages of pesticides increased by 19-38% under ultrasound treatment. Ultrasonic intensity, pulse duty cycle, and duration time were key factors affecting microbial growth and pesticide degradation. Under the optimal ultrasonic pre-treatment conditions, the degradation rate constants of four pesticides were at least 3.4 times higher than those without sonication. In addition, such ultrasound pretreatment significantly shortened the yogurt fermentation time, increased the water holding capacity, hardness and antioxidant activity of the yogurt, and improved the flavor quality of the yogurt. Conclusion: Ultrasonic pretreatment significantly accelerated the degradation of the four pesticides during yogurt fermentation. In addition, such ultrasound pretreatment increased the efficiency of yogurt making and improved the quality of yogurt in terms of water holding capacity, firmness, antioxidant activity and flavor. These findings provide a basis for the application of ultrasound to the removal of pesticide residues and quality improvement of yogurt. This article is protected by copyright. All rights reserved.
... In the context of productivity enhancement in bioprocess intensification, ultrasound is generally limited to low power inputs to limit biocatalyst damage whilst still improving the mixing and mass transfer rates of the process. Much research effort has been directed at studying the effects of ultrasound and its cavitation impacts on whole cell fermentations and enzymatic reactions, many of which are discussed in detail in recently published reviews on these subjects [190][191][192][193][194]. A few pertinent and recent examples will be highlighted here to demonstrate the extent of intensification that can be realised by the ultrasound technique in bioprocessing. ...
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With the current pressing need to rise to the ambition of net zero targets to mitigate carbon emissions and climate change impacts, sustainable processing has never been more critical. Bioprocessing has all the desirable attributes to respond to the sustainable processing challenge: use of cheap, renewable resources, nature-inspired, highly selective biocatalysts operating optimally under mild conditions and reduced energy consumption/carbon footprint. With bioprocessing productivity being far from ideal to meet the large-scale need for food, drugs, biofuels and bio-based chemicals, there has been tremendous interest of late in developing intensified bioprocesses, with significant advancement achieved in tailoring and utilising the technologies in the toolbox traditionally applied in chemical process intensification. This review highlights the wide range of activities currently on-going in bioprocess intensification, focusing on upstream, bioreactor/fermentation and downstream separation steps. Great strides have been made in biocatalyst engineering and high density cell immobilisation for significant productivity enhancement, which, in conjunction with elegant process innovations such as novel bioreactor technologies and in-situ product separations, are enabling bioprocesses to become more competitive than ever before. The future prospects of bioprocess intensification are promising but there are still challenges that need to be overcome to fully exploit this technology.
... During cavitation, the microjets formed increase the cell porosity and helps to transfer the oxygen to interior cells enhancing the mass transfer [156]. Under these conditions, ultrasound enhances the growth of microorganisms due to increased cell membrane permeability for substrates, gases, and other nutrient components [157]. The mass transfer effects generated due to acoustic cavitation could also be used to simply enhance the rates of chemical reactions or chemical processes such as dairy fermentation. ...
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Alternative methods for improving traditional food processing have increased in the last decades. Additionally, the development of novel dairy products is gaining importance due to an increased consumer demand for palatable, healthy, and minimally processed products. Ultrasonic processing or sonication is a promising alternative technology in the food industry as it has potential to improve the technological and functional properties of milk and dairy products. This review presents a detailed summary of the latest research on the impact of high-intensity ultrasound techniques in dairy processing. It explores the ways in which ultrasound has been employed to enhance milk properties and processes of interest to the dairy industry, such as homogenization, emulsification, yogurt and fermented beverages production, and food safety. Special emphasis has been given to ultrasonic effects on milk components; fermentation and spoilage by microorganisms; and the technological, functional, and sensory properties of dairy foods. Several current and potential applications of ultrasound as a processing technique in milk applications are also discussed in this review.
... Industrial and experimental applications of sonochemistry have been proven in nanotechnology, biotechnology and medicine [23,24]. Ultrasound-assisted techniques have been rapidly developed for a variety of chemical processes [25]. ...
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Sonication of implants has been shown to be a promising method for diagnosis of prosthetic infections due to its improved sensitivity, simplicity, and low cost. The aim of the present study was to evaluate the effects of ultrasound performed under different conditions regarding temperature, duration, and composition of sonication tubes on bacterial species often associated with prosthetic infections. We found that ultrasound had an inhibitory effect on bacteria, of which gram-negative bacteria, in particular Escherichia coli, were almost eradicated after 5 min of sonication at 35°C. Gram-positive bacteria were found to be resistant to the effect of ultrasound. Four factors were important for the inhibitory effect of sonication: the type of microorganism, the temperature of the sonication buffer, the duration of exposure to ultrasound (minutes), and the material and composition of the sonication tube in which sonication is performed. On the basis of the results from the present study, we propose a protocol for sonication and recovery of bacteria associated with biofilm on infected implants prior to conventional culture. From the present protocol, we recommend sonication for 7 min at 22°C at the maximum effect which permits survival of gram-negative bacteria.
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Some species of bacteria produce colonies and spores which agglomerate in spherical clusters (Bacillus subtilis) and this serves as a protection for the organisms inside against biocidal attack. Flocs of fine particles e.g. clay can entrap bacteria which can also protect them against the biocides. It is because of problems such as these that alternative methods of disinfecting water are under active investigation. One such method is the use of power ultrasound, either alone or in combination with other methods. Ultrasound is able to inactivate bacteria and deagglomerate bacterial clusters or flocs through a number of physical, mechanical and chemical effects arising from acoustic cavitation. The aim of this study was to investigate the effect of power ultrasound at different powers and frequencies on Bacillus subtilis. Viable plate count techniques were used as a measure of microbial activity. Results showed a significant increase in percent kill for Bacillus species with increasing duration of exposure and intensity of ultrasound in the low-kilohertz range (20 and 38 kHz). Results obtained at two higher frequencies (512 and 850 kHz) indicated a significant increase in bacteria count suggesting declumping. In assessing the bacterial kill with time under different sonication regimes three types of behaviour were characterized: High power ultrasound (lower frequencies) in low volumes of bacterial suspension results in a continuous reduction in bacterial cell numbers i.e. the kill rate predominates. High power ultrasound (lower frequencies) in larger volumes results in an initial rise in cell numbers suggesting declumping of the bacteria but this initial rise then falls as the declumping finishes and the kill rate becomes more important. Low intensity ultrasound (higher frequencies) gives an initial rise in cell numbers as a result of declumping. The kill rate is low and so there is no significant subsequent decrease in bacterial cell numbers.
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We report in this study that the sonication of laccase from Trametes villosa and bovine serum albumin promotes the formation of protein aggregates with high molecular weight. The formation of aggregates leads to the deactivation of the enzyme, fact that was confirmed by the analysis of the enzyme stability (half-life time) upon ultrasound treatment. This inactivation was mainly caused by the radicals formed by the cavitation phenomenon. It was verified that the addition of polyvinyl alcohol to laccase had a protecting effect against enzyme inactivation. The performance of laccase in the decolourization of indigo carmine was studied. It was observed that the best results were attained when the dye solution was treated with ultrasound and enzyme stabilized with polyvinyl alcohol, where more than 65% of decolourization was achieved. This value is remarkably higher than that attained for the enzyme alone, which was only able to decolourize 20% of the dye solution within 1h of treatment. These results have important implications for the exploitation of sonication in textile industry, where the pollution caused by the release of dyes into effluents is one of the major concerns.
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The enhanced β-glucosidase of Lactobacillus plantarum BCRC 10357 was investigated by a strategic ultrasound treatment on bacteria at different growth stages to induce biological stress response for biotransformation of isoflavones from glucosides to aglycones in okara. Without ultrasound, the highest β-glucosidase activity was occurred in 12-h incubation as 24.45 U/mL; while after ultrasound treatment at death phase, the highest β-glucosidase activity was greatly enhanced to 48.80 U/mL in 24-h re-incubation, showing the bacteria at early death phase behaving the highest resistance to stress than at other stages. With strategically ultrasound-treated L. plantarum BCRC 10357 to ferment okara in the broth at 37 °C, the fraction of bioactive aglycones (daidzein and genistein) in isoflavones was increased from 57% in 12 h to 89% in 36 h. The effective method to improve β-glucosidase released from L. plantarum was developed for the valorization of okara as potential functional ingredients in food.
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Fermentation processes involve the participation of enzymes and organic catalysts, generated by range of microorganisms, to produce chemical transformations. Ultrasound can be used in such processes to either monitor the progress of fermentation or to influence its progress. High frequency ultrasound (> 2MHz) has been extensively reported as a tool for the measurement of the changes in chemical composition during fermentation providing real time information on reaction progress. Low frequency ultrasound (20 – 50 kHz) can influence the course of fermentation by improving mass transfer and cell permeability leading to improved process efficiency and production rates. It can also be used to eliminate micro-organisms which might otherwise hinder the process. This review summarises key applications of high and low frequency ultrasound in food fermentation applications.
Article
The effect of a ‘bath type’ ultrasound on the fermentation of beer made from Korean six-row barley was studied. Beer samples were treated in an ultrasonication bath for 4 days during primary fermentation. The frequency of the ultrasound was 40 kHz, and the input power was adjusted to 120, 160 and 200 W. Ultrasonic treatment was performed for 2, 6 and 12 h for each input power. The physicochemical and sensory properties, as well as the quality of the beers were measured. Ultrasonication enhanced ethanol production by 13.18% at 160 W. Copyright © 2015 The Institute of Brewing & Distilling
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This chapter first addresses the emergence of ultrasonic cleaning technology. The principles and mechanism of application are discussed, and an explanation of the benefits of ultrasonic cleaning is provided. Ultrasonic cleaning also has limitations, which are revealed, along with reasons for these shortfalls. This chapter serves as a practical guide for maximizing and monitoring the effectiveness of an ultrasonic cleaning system. Advances in ultrasonic technology are explored along with their impetus. Finally, this chapter speculates on the future of ultrasonic cleaning. An appendix on recent developments in ultrasonic washing of textiles also is included.
Article
Objectives: The aim of this study was to obtain valuable information about the effect of ultrasonic irradiation with a frequency of 30 kHz frequency and power of 100 W on the inactivation capability of two bacterial groups, namely, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, in physiologic water samples. Methods: Ultrasonic irradiation of bacterial samples with different populations of 5 × 10(3), 1.5 × 10(4), and 3 × 10(4) colony-forming units/mL was performed at a constant frequency with various treatment times. The specific energy (γ) values were calculated for these different concentrations of E coli and S aureus. The rate constant for ultrasonic inactivation was estimated in the linear region of a plot representing a survival ratio logarithm versus sonication time. Results: Although a significant death rate for E coli was observed with ultrasound treatment, in contrary to expectations, an increase in S aureus populations was observed. Conclusions: Considering the widespread use of ultrasound for sterilization of tools and equipment used in hospitals, the results obtained in this study indicate that ultrasonic irradiation is not a suitable method for the elimination of the major hospital pathogen S aureus.
Article
This paper presents a study of low-ultrasonic energy that can be affected to increase yeast growth. Forces of the ultrasonic stimulation in the yeast media broth, a liquid nutrient medium for the culture of yeast, produce change of liquid densities and surrounding pressure, which affect to the yeast growth. A utilized ultrasonic system including transducer of 20 kHz was varied powers and applied into the 200 ml. starter broth including Saccharomyces cerevisiae yeast. Those powers can be adjusted intensities, repetition times and durations to stimulate yeast cell. For monitoring the yeast growth, a cell direct-count with a Hemacytometer and an optical density using a Spectrophotometer were employed every 1 hour. Experiment results were shown enhancements of the yeast growth which were stimulated by the various ultrasonic powers compared with commonly used methods. Consequently, the ultrasonic can be applied to accelerate the yeast growth in fermentation to increase productivity and decrease the fermentation time.
Article
The present endeavor encompasses process intensification approach for daunorubicin fermentation from Streptomyces peucetius MTCC 4332 using ultrasonic stimulus. One factor at a time design was employed to illustrate the effect of various process parameters of ultrasonic irradiation (irradiation at various growth phases, power, irradiation time and duty cycle) on enhancement of daunorubicin productivity. Ultrasonic irradiation on 4th day of bacterial culture growth at 25 kHz frequency with 160 W power and 40% duty cycle for 5 min shows 1.62 fold increase in daunorubicin production (from 46.96 to 76.42 mg/L) as compared to non sonicated batch fermentation. Higher duty cycle imparts detrimental effect on bacterial cell and decreases viability of cells. Environmental scanning electron microscopic analysis depicted structural integrity of bacterial cell after irradiation of optimized ultrasonic dose. Moreover, declined glucose concentration after ultrasonication from 1.16 to 0.46 gm/L after 7 days of incubation, implies that cellular uptake of substrate was increased and productivity of cell was also enhanced. Hence, proposed process intensification approach will be very useful to boost up the fermentation of various therapeutic molecules
Article
A potent fibrinolytic protease (thrombinase) was isolated from marine actinomycetes which can be used for the treatment of myocardial infarction. Thrombinase producing actinomycetes were isolated from the marine water sample using fibrin plate assay. The spore morphology of the marine isolate was analyzed by light and scanning electron microscopy (SEM). Further the marine isolate was identified as Streptomyces venezuelae through biochemical tests and 16srRNA analysis. Effect of ultrasonication on cell concentration and enzyme activity was further determined and the maximum values 20.62 mg mL−1 and 6.24 FU mL−1, respectively were obtained under static condition. The maximum specific growth rate μm (h−1) of the marine isolate under static condition was evaluated as 0.554 h−1 with ultrasonication by modified gompertz model using Matlab 7.0. Moreover effect of salinity on cell concentration and enzyme activity was also analyzed, the maximum biomass, 22.42 mg mL−1 and maximum enzyme activity, 6.7 FU mL−1 at 6% (w/v) salinity. Optimization was done further by response surface methodology (RSM) using central composite design (CCD) and it was found pH 7, temperature 35 °C and salinity 6% (w/v) as optimum for maximum specific growth rate, 0.578 h−1 and thrombinase activity, 8.34 FU mL−1.
Article
The effects of ultrasonic wave irradiation on cell growth and the formation of ethanol and other volatile components in the fermentation process were investigated. The fermentation periods were reduced to 50–64% in wine, beer, and sake made from saccharified rice solution when weak ultrasonic waves were irradiated at 30 mW/cm2; the total intensity was 590 mW. YEPD medium fermented by a combination of optimal thermal trajectory control and continuous ultrasonic irradiation yielded a concentration of isoamylacetate about 2.5 times greater than the maximum concentration under isothermal conditions (20°C). Irradiation had virtually no effect when the dissolved carbon dioxide concentration (DCO2) was maintained at a level lower than the experimental value of Bunsen's CO2 absorption coefficient. Therefore, it is suggested that irradiation accelerates the formation of ethanol and other components mainly by decreasing DCO2.
Article
Microstructure changes and micro-damage behavior of some LAB under the impact of ultrasound shock (20 kHz for 5, 10, 15, 20, 25 and 30 minutes) was studied by optical microscope and Transmission Electron Microscope (TEM). Three kinds of micro damages are usually produced by ultrasound, micro-cracks, micro-voids and ruptures.Studies, by TEM showed that ultrasound can increase the cell wall permeability of the cells, which is important in the release of enzymes such as ß-galactosidas reduction of the coagulation time. The survival of LAB was very low in very long exposures of ultrasound.
Article
Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This paper attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3-propanediol (1,3-PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3-PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters we attribute these results to increased substrate-enzyme affinity and decreased substrate inhibition for 1,3-PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3-PDO. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
Article
We investigate the stability and rupture kinetics of planar lipid membranes covered with electrostatically adsorbed polyelectrolytes. After black lipid membranes were formed from negatively charged lipids, polylysines (PLs) of different molecular weights (MW) were added on one or on both sides of the membrane. The adsorption of PL was detected by recording changes of the transmembrane voltage. Rupture was induced by applying short voltage pulses across the membrane. The voltage causing breakdown of the membrane gives information on its mechanical stability. Adsorption of PL on one side of the membrane leads to an asymmetric transmembrane potential, which adds to the externally applied voltage. High MW PL decreases the critical breakdown voltage of the membrane significantly but also increases the delay time between the voltage pulse and pore formation. It is further shown that PL alters the time course of pore widening in a molecular weight-dependent manner. Low MW PL-decorated membranes and undecorated membranes show a fast rupture determined by inertia. In contrast, adsorption of high MW PLs causes a dramatic decrease of the rupture velocity. In this case, the rupture velocity is determined by viscosity. An analysis of the rupture kinetics allows an estimate of the 2D viscosity.
Article
With significant growth of biodiesel industry in past few years, very large quantities of crude glycerol are available as side product of transesterification process. Effective conversion of glycerol to value added products can boost economy of biodiesel industry. The present study attempts to investigate ultrasound assisted bioconversion of glycerol to 1,3-propanediol (1,3-PDO) and ethanol (EtOH) from mechanistic. A dual approach of coupling experiments under ultrasound irradiation or mechanical shaking with simulations of cavitation bubble dynamics has been adopted. Effect of ultrasound on bioconversion has been assessed with the help of kinetic analysis of enzymatic reactions. The nature of convection generated by ultrasound irradiation at raised static or ambient pressure has dual character of oscillatory motion of liquid, accompanied by medium amplitude jerks by the acoustic or shock waves emitted by cavitation bubbles, which could contribute towards enhancement of the enzymatic reactions in metabolic pathway. In presence of ultrasound, the highest yield (0.06 mol/mol) of 1,3-PDO was obtained with lowest glycerol concentration (5 g/L) while highest ethanol yield (0.12 mol/mol) was observed at 10 g/L glycerol concentration. Yield of both products increases under ultrasound irradiation, but lesser enhancement is seen for 1,3 PDO than EtOH due to substrate inhibition. Maximum enhancement in yield (49%) of 1,3-PDO was seen for 10 g/L glycerol concentration. The role of ultrasound irradiation in enhancement of bioconversion of glycerol is found to be of physical nature, without any influence on biochemistry of glycerol conversion.
Article
Reducing production costs for fermentation-based drugs (e.g., antibiotics) is crucial for the long-term sustainability of healthcare. In this study, we propose a novel low-intensity pulsed ultrasound (LIPUS) stimulation scheme using a nominal frequency of 1.5 MHz with a 20% duty cycle (200 μs ultrasound on and 800 μs ultrasound off) to increase production of fermentation-based drugs. We chose Penicillium brevicompactum as a model system to demonstrate the performance of our LIPUS system. Penicillium brevicompactum can produce mycophenolic acid (MPA), an immunosuppressive agent commonly used to prevent rejection after organ transplantation. We have stimulated Penicillium brevicompactum in 50 mL shake flasks using LIPUS during its fermentation. After a series of screening experiments to optimize ultrasound parameters (e.g., ultrasound intensities, treatment duration and treatment frequency per day), it was concluded that ultrasound stimulation can increase MPA production by as much as 60% when treated eight times a day for 10-min durations at an intensity (spatial peak temporal average) of 200 mW/cm(2). These findings elucidate a new approach to reduce the cost of producing fermentation-based drugs.
Article
BACKGROUND: Fermentations of Aspergillus terreus are commercially used to produce lovastatin. How ultrasound might influence this fermentation is unknown. While high-intensity ultrasound is effective in disrupting microbial cells, ultrasound of low intensity is known to improve productivity of some fermentation processes without damaging cells. Mechanisms behind productivity improvements have not been clearly identified in earlier studies. This work reports on the effects of ultrasound on A. terreus fermentation for low (957 W m−3), medium (2870 W m−3) and high (4783 W m−3) values of sonication power input in a slurry bubble column sonobioreactor. RESULTS: Sonication at any power level did not affect biomass growth profiles in comparison with negative controls. In contrast, medium- and high-intensity sonication greatly reduced production of lovastatin and substantially altered the growth morphology. At medium and high intensity, ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Sonication affected broth rheology because rheology depends on the morphology of the suspended biomass. CONCLUSION: Sonication can be used to modify growth morphology and broth rheology without affecting growth of filamentous fungi. Sonication appears to influence the primary growth metabolism and secondary metabolism differently in different situations. Copyright
Article
Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.
Article
Cavitation results in the generation of hot spots, highly reactive free radicals, and turbulence associated with liquid circulation currents, which can result in the intensification of various physical/chemical operations. The present work provides an overview of the applications of the cavitation phenomenon in the specific area of biochemical engineering/biotechnology, discussing the areas of application, the role of cavitation, the observed enhancement and its causes by highlighting some typical examples. The different methods of inducing cavitation and the dominance of one over the other, mostly with respect to energy requirements, in different areas of biotechnological application are discussed. The major applications discussed in the work include microbial cell disruption for the release or extraction of enzymes, microbial disinfection, wastewater treatment, crystallization, synthesis of biodiesel, emulsification, extraction of bio-components, freezing and gene transfer into cells or tissues. Some recommendations for optimal operating/geometric parameters have also been made. Overall, it appears that the combined efforts of physicists, chemists, biologists and chemical engineers are required to effectively use cavitational reactors for industrial applications.
Article
The effects of ultrasound on the activities of alpha-amylase and amyloglucosidase toward starch and glycogen hydrolysis, and of invertase toward sucrose hydrolysis, were investigated using an ultrasound bath. A fixed-time assay was employed with product formation determined by reducing equivalents formed per unit time using the 3,5-dinitrosalicylate reagent (DNS). Reaction rates in the presence and absence of ultrasound were compared. Increases in rates were observed in the presence of ultrasound at most concentrations with a marked increase in invertase activity towards sucrose (37% at 0.9 m) and a reduction in the inhibition of sucrose hydrolysis observed at high substrate concentrations. Improvements in the efficiency of mixing and disruption of intra- and intermolecular substrate molecule interactions at high concentrations are possible explanations for the changes observed.
Article
Sonolysis, enzyme treatment, and a combination of the two processes were tested for the degradation of phenol in aqueous medium. With sonolysis alone, 423 kHz ultrasound eliminated phenol molecules through hydroxyl radical reactions. In the presence of hydrogen peroxide, the enzyme horseradish peroxidase (HRP) catalyzed the oxidation of phenol to insoluble polymers which could be separated easily from the solution. In the combined system (HRP, H2O2, US-423 kHz) the degradation rate was accelerated, and the color of the solution changed to dark brown without any precipitate formation. The enhancement was more significant in the presence of additives such as polyethylene glycol (PEG). The higher rate induced through the use of ultrasound leads to the reduction of HRP dose requirements, which is of importance for the economic feasibility of this method. The enzyme solution alone did not show any inactivation up to 45 min sonication at 423 kHz. Investigation of phenol removal efficiency was mainly focused on enzyme, phenol and PEG concentrations, as well as the sequence and method of addition of reagents.
Article
Production of ethanol from lactose by fermentation with the yeast Kluyveromyces marxianus (ATCC 46537) under various sonication regimens is reported. Batch fermentations were carried out at low-intensity sonication (11.8W cm(-2) sonication intensity at the sonotrode tip) using 10%, 20% and 40% duty cycles. (A duty cycle of 10%, for example, was equivalent to sonication for 1 s followed by a rest period (no sonication) of 10s.) Fermentations were carried out in a 7.5 L (3 L working volume) stirred bioreactor. The sonotrode was mounted in an external chamber and the fermentation broth was continuously recirculated between the bioreactor and the sonication chamber. The flow rate through the sonication loop was 0.2 L min(-1). All duty cycles tested improved ethanol production relative to control (no sonication). A 20% duty cycle appeared to be optimal. With this cycle, a final ethanol concentration of 5.20 +/- 0.68 g L(-1) was obtained, or nearly 3.5-fold that of the control fermentation. Sonication at 10% and 20% cycles appeared to stimulate yeast growth compared to the control fermentation, but 40% duty cycle had a measureable adverse impact on cell growth. Sonication at 10% and 20% cycles enhanced both the extracellular and the intracellular levels of beta-galactosidase enzyme. Although at the highest duty cycle sonication reduced cell growth, cell viability remained at >= 70% during most of the fermentation. Sonication at a controlled temperature can be used to substantially enhance productivity of bioethanol fermentations.
Article
Ultrasound (US) has become a ubiquitous technological process in a large variety of scientific disciplines. However, little information exists on the use of ultrasound to enhance biological processes and/or processing and consequently this paper provides an overview of work reported to date on this topic. This review provides a brief introduction to ultrasound and the history of ultrasound as applied to bioprocesses. This is followed by a discussion of the influence of US on discrete enzyme systems, enzymes used in bioremediation, microbial fermentations and enzymatic hydrolysis of biopolymers. Augmentation of anaerobic digestion by US is then considered along with enhancement of enzymes in food science and technology. The use of ultrasonically stimulated enzymes in synthesis is then considered and other relevant miscellaneous topics are described. It is concluded that the precise mechanism of action of US in bio-processing remains to be elucidated though a variety of plausible suggestions are made.
Article
Aims: This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro-organisms (Escherichia coli and Klebsiella pneumonia). Methods and Results: Two analytical methods were employed; viable plate counts (CFU ml−1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions: Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and Impact of the study: Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.
Article
This paper presents an analysis of lag phase phenomena in Saccharomyces cerevisiae growth as a function of ultrasonic irradiation. Pulse irradiation treatments were performed by a 20 kHz ultrasonic transducer with different durations and energies. Data obtained from experiments were then employed to estimate growth parameters by specific transfer function. The significance of the different lag times in response to ultrasonic irradiation was analysed. The results showed that the yeast growth in lag phase responded to the irradiated ultrasonic of 20 min more than the 10 min. The ultrasonic energies between 330 and 360 W s m(-3) could decrease lag time up to 1 h compared to the sample without ultrasonic irradiation. Conversely, the treatments with energies higher than 850 W s m(-3) were able to extend the lag time and decrease the yeast growth. The lag durations of S. cerevisiae were changed significantly by different ultrasonic irradiations, energies and durations. In particular, sufficient irradiation energies reduced the lag time, resulting in accelerated yeast growth. In contrast, high energy could inactivate growth by increasing the lag time. Significance and Impact of the Study:  This work provides an alternative technique to either accelerate or inactivate the S.cerevisiae lag phase. The approach can be developed in experiment designed to control the yeast growth by ultrasonic irradiation as assistance in the environments.
Article
A non-woven titanium dioxide (TiO(2)) fabric was applied to disinfection by ultrasound (US) irradiation, and the disinfection efficiency and lipid peroxidation of Escherichia coli (E. coli) cell membrane were evaluated to investigate the killing process. The addition of non-woven TiO(2) fabric enhanced hydroxyl (OH) radical generation and disinfection efficiency. Judging from the disinfection experiments using glutathione or t-butanol as a radical scavenger, the OH radical played a major role in cell killing in sonodynamic disinfection with non-woven TiO(2) fabric. Moreover, to understand the detailed killing process, damage to cell membrane was also evaluated using a diphenyl-1-pyrenylphosphine (DPPP) fluorescent probe, which detects the membrane's lipid peroxidation. The addition of non-woven TiO(2) fabric aggravated this peroxidation. This aggravation was caused by the OH radical according to an assay using a radical scavenger. From these results, it was concluded that non-woven TiO(2) fabric as a sonocatalyst promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced a major disorder in the E. coli cell membrane under US irradiation.
Article
The chemical effects of ultrasound in aqueous solutions are due to acoustic cavitation, which refers to the formation, growth, and collapse of small gas bubbles in liquids. The very high temperatures (several thousand K) and pressures (several hundred atmospheres) of collapsing gas bubbles lead to the thermal dissociation of water vapor into .OH radicals and .H atoms. Their formation has been confirmed by electron spin resonance (ESR) and spin trapping. The sonochemistry of aqueous solutions of gases and of volatile and nonvolatile solutes is reviewed. The similarities and differences between sonochemistry and radiation chemistry of aqueous solutions are explained. Some unusual characteristics of aqueous sonochemistry can be understood by considering the properties of supercritical water. By the use of rare gases with different thermal conductivities, it is possible to distinguish between temperature-dependent processes such as redox reactions initiated by .OH radicals and .H atoms and pressure-dependent processes which lead to polymer degradation and cell lysis. The evidence for free radical formation in aqueous solutions by pulsed ultrasound is discussed. This subject is of interest because it is related to the possible deleterious effects of ultrasonic diagnostic devices. The role of free radicals and of mechanical effects induced by ultrasound in DNA degradation, inactivation of enzymes, lipid peroxidation, and cell killing is reviewed.
Article
The commercial production of ethanol from cellulose by simultaneous saccharification and fermentation (SSF) is prevented in part by the high cost of fungal cellulase enzymes. Intermittent exposure of SSF processes to ultrasonic energy under selected conditions (5 FPU of cellulase/g of substrate; 15 min of exposure/240 min cycle during the latter half of SSF) was found to increase ethanol production from mixed waste office paper by approximately 20%, producing 36.6 g/L ethanol after 96 h (70% of the maximum theoretical yield). Without ultrasound, 10 FPU of cellulase/g of substrate was required to achieve similar results. Continuous exposure of the organism to ultrasonic energy was bacteriostatic and decreased ethanol production but may be useful for the controlling bacterial growth in other processes.
Article
Enhanced metabolic productivity of microbial, plant and animal cells in bioreactors can greatly improve the economics of biotechnology processes. Ultrasound is one method of intensifying the performance of live biocatalysts. Ultrasonication is generally associated with damage to cells but evidence is emerging for beneficial effects of controlled sonication on conversions catalyzed by live cells. This review focuses on the productivity enhancing effects of ultrasound on live biological systems and the design considerations for sonobioreactors required for ultrasound-enhanced biocatalysis.
Article
Ultrasound was employed to increase the growth rate of bacterial cells attached to surfaces. Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli cells adhered to and grew on a polyethylene surface in the presence of ultrasound. It was found that low-frequency ultrasound (70 kHz) of low acoustic intensity (<2 W/cm(2)) increased the growth rate of the cells compared to growth without ultrasound. However, at high intensity levels, cells were partially removed from the surface. Ultrasound also enhanced planktonic growth of S. epidermidis and other planktonic bacteria. It is hypothesized that ultrasound increases the rate of transport of oxygen and nutrients to the cells and increases the rate of transport of waste products away from the cells, thus enhancing their growth.
Article
The structure of the cell membrane of Ecemothecium ashbyii is considered to be one of the main obstacles in the improvement of riboflavin productivity, which impedes the release of riboflavin from the cell into the fermentation broth. The results of the present study show that stimulation on growth and riboflavin biosynthesis phase, the content of riboflavin was most obviously enhanced, as compared with the control and the total riboflavin quantity released by ultrasonic treatment. The optimal stimulation time was from 104 to 112 h and ultrasonic should be loaded every 1.5 h.
Article
Gas-filled microbubbles have been successfully used as gene delivery reagents in combination with diagnostic ultrasound. Although shock wave exposure has been shown to transfect cells with naked DNA in vitro, it has not been tested whether the addition of microbubbles would augment DNA uptake under those conditions. Therefore, the aim of this study was to test the impact of microbubbles on transgene expression in vitro under shock wave exposure conditions. HEK 293 cells were treated with 60 or 120 pulses of shock waves at varying energy levels. Cells were mixed with either 100 μg/mL luciferase expressing plasmid DNA or with microbubbles that were produced with the same amount of this DNA. Cell death was evaluated after 1 h and transgene expression, after 24 h. In the presence of microbubbles, transgene expression was significantly higher (as much as 29-fold) relative to that obtained without microbubbles. Cells exposed to 120 pulses demonstrated higher transgene expression (as high as 2.7-fold) compared with cells exposed to 60 pulses. The use of microbubbles resulted in greater cell death, varying from 26% (low energy) to 78% (high energy). In conclusion, DNA-loaded microbubbles can significantly increase shock wave mediated gene transfer. However, this effect is associated with increased levels of cell destruction. (E-mail: [email protected] /* */).