Purification of serratiopeptidase from Serratia marcescens NRRL B 23112 using ultrasound assisted three phase partitioning

The ultrasound assisted three phase partitioning (UATPP) is a novel bioseparation method for separation and purification of biomolecules. In the present work, UATPP was investigated for the first time for purification of serratiopeptidase from Serratia marcescens NRRL B 23112. Effect of various process parameters such as ammonium sulphate saturation, t-butanol to crude extract ratio, pH, ultrasonic frequency, ultrasonic intensity, duty cycle and irradiation time were evaluated and optimized. The optimized conditions were found to be as follows: ammonium sulphate saturation 30% (w/v), pH 7.0, t-butanol to crude ratio 1:1 (v/v), ultrasound frequency 25 kHz, ultrasound intensity 0.05 W/cm², duty cycle 20% and irradiation time 5 min. The maximum purity and recovery obtained from UATPP was 9.4-fold and 96% respectively as compared to the three phase partitioning (TPP) (4.2-fold and 83%). Also the process time for UATPP was significantly reduced to 5 min from 1 h as compared to TPP. The results indicate that, UATPP is an efficient technique for the purification of serratiopeptidase with maximum purity, recovery and reduced processing time.     

Ultrasound stimulated production of a fibrinolytic enzyme

The present study is aimed at enhanced production of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672 under ultrasonic stimulation. Various process parameters viz; irradiation at different growth phases, ultrasonication power, irradiation duration, duty cycle and multiple irradiation were studied for enhancement of fibrinolytic enzyme productivity. The optimum conditions were found as follows, irradiation of ultrasonic waves to fermentation broth at 12 h of growth phase with 25 kHz frequency, 160 W ultrasound power, 20% duty cycle for 5 min. The productivity of fibrinolytic enzyme was increased 1.82-fold from 110 to 201 U/mL compared with the non sonicated control fermentation. Drop in glucose concentration from 0.41% to 0.12% w/v in ultrasonicated batch implies that, ultrasonication increases the cell permeability, improves substrate intake and progresses metabolism of microbial cell. Microscopic images before and after ultrasonic stimulation clearly signifies the impact of duty cycle on decreasing biomass concentration. However, environmental scanning electron micrograph does not show any cell lysis at optimum ultrasonic irradiation. Offshoots of our results will contribute to fulfill the demand of enhancement of microbial therapeutic enzyme productivity, through ultrasonication stimulation.     

Ultrasound assisted enzymatic pre-treatment of high fat content dairy wastewater

This paper illustrates the application of ultrasound in a dairy waste water treatment for the removal of fat using enzyme as a catalyst. Lipase Z was used to perform the enzymatic pre-hydrolysis of a synthetic dairy wastewater containing around 2000 mg/L of fat content coupled with ultrasound irradiation. Different process parameters like effect of enzyme loading, temperature, ultrasound power, frequency, duty cycle and speed of agitation are optimized. The maximum hydrolysis of 78% is achieved at 0.2% enzyme loading (w/v), 30 °C temperature, 165 W of ultrasonication power at 25 kHz and 66% duty cycle. It was observed that the enzymatic pre-hydrolysis under the influence of ultrasound drastically reduces the reaction time from 24 h to 40 min as compared to conventional stirring with improved yield.     

Sonochemical synthesis of hierarchical ZnO nanostructures

This work is about fabrication of ZnO nanostructures (ZnO-NS) via a simple sonochemical method. The chemicals used for the synthesis of various shaped ZnO are Zn salt, sodium hydroxide and ammonia solution without other structure directing agent or surfactant needed. This method is feasible and green, as it does not require high temperature and/or highly toxic chemicals. The shape of the ZnO-NS can be tuned by adjusting the ultrasound energy dissipated via varying the ultrasonication time from 5 to 60 min. It was found that uniform ZnO nanorods with diameter around 50 nm were formed after 15 min of ultrasonication while flowerlike ZnO-NS was formed after 30 min. This method produces high quality ZnO-NS with controllable shapes, uniformity, and purity.     

Ultrasound assisted intensified recovery of lactose from whey based on antisolvent crystallization

The current work deals with understanding the fundamental aspects of intensified recovery of lactose from paneer (cottage cheese) whey using the anti-solvent induced sonocrystallization. Ultrasonic horn (22 kHz) with varying power levels over the range of 40–120 W has been used for initial experiments at 100% duty cycle and two different levels of ultrasonic exposure time as 10 min and 20 min. Similar experiments were also performed using ultrasonic bath for the same time of exposure but with at two ultrasonic frequencies (22 kHz and 33 kHz). It was observed that the lactose recovery as well as purity increased with an increase in ultrasonic power at 100% duty cycle for the case of treatment time as 10 min whereas the lactose recovery and purity increased only till an optimum power for the 20 min treatment. In the case of ultrasonic bath, lactose purity increased with an increase in the ultrasonic frequency from 22 kHz to 33 kHz though the lactose recovery marginally decreased. Overall, it was observed that the maximum lactose recovery was ∼98% obtained using ultrasonic horn while the maximum lactose purity was ∼97%. It was also observed that maximum lactose recovery was ∼94% for the case of ultrasonic bath while the maximum lactose purity was ∼92%. The work has enabled to understand the optimized application of ultrasound so as to maximize both the lactose yield and purity during the recovery from whey.     

Aqueous two-phase system coupled with ultrasound for the extraction of lignans from seeds of Schisandra chinensis (turcz.) Baill

In this study the potential use of an aqueous two phase system (ATPS) coupled with ultrasound for the extraction of lignans from Schisandra chinensis seeds was evaluated and optimized using response surface methodology (RSM). The main bioactive components, schizandrin (SA), schisantherin A (SAA) and deoxyschizandrin (DSA) were selected as markers. The partitioning behavior of lignans in different salt-types of ATPS was compared. The optimization ATPS of 25% (w/w) (NH₄)₂SO₄ and 19% (w/w) ethanol were selected based on their higher upper phase partitioning coefficient (>74) and the recovery (>93%) for three markers. Using the optimized ATPS solvent, the RMS results showed 20:1 of solvent:solid, 800 W and 61.1 min were the optimal ultrasound assisted extraction conditions, under which 13.10 mg/g SA, 1.87 mg/g SAA and 1.84 mg/g DSA were recovered in the upper phase, whereas the wasted stigmas accumulated in the lower phase. Compared with 80% ethanol (v/v) ultrasonic extraction, similar yields were obtained, but the present method exhibited higher extraction purity for the selective extraction of lignans from S. chinensis seeds.     

Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound

The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20 kHz) and high-frequency (850 kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50 W for 60 min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the κ-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of α-lactalbumin and β-lactoglobulin slightly decreasing.     

Extraction mechanism of ultrasound assisted extraction and its effect on higher yielding and purity of artemisinin crystals from Artemisia annua L. leaves

This study proposes an ultrasound-horn system for the extraction of a natural active compound “artemisinin” from Artemisia annua L. leaves as an alternative to hot maceration technique. Ultrasound leaching improves artemisinin recovery at all temperatures where only ten minutes is required to recover 70% (4.42 mg g⁻¹) compared to 60 min of conventional hot leaching for the same yield. For instance, ultrasound treatment at 30 °C produced a higher yield than the one obtained by conventional maceration at 40 °C. Kinetic study suggests that the extraction pattern can be assimilated, during the first ten minutes, to a first order steady state, from which activation energy calculations revealed that each gram of artemisinin required 7.38 kJ in ultrasound versus 10.3 kJ in the conventional system. Modeling results indicate the presence of two extraction stages, a faster stage with a diffusion coefficient of 19 × 10⁻⁵ cm² min⁻¹ for ultrasound technique at 40 °C, seven times higher than the conventional one; and a second deceleration stage similar for both techniques with diffusion coefficient ranging from 1.7 to 3.1 × 10⁻⁵ cm² min⁻¹. It is noted that the efficient ultrasound extraction potential implies extraction of higher amount of co-metabolites so low artemisinin crystal purity is engendered but a combination with a purification step using activated charcoal and celite adsorbents produced crystals with comparable purity for conventional and ultrasound samples.     

Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition

The present work demonstrates the hydrolysis of waste cooking oil (WCO) under solvent free condition using commercial available immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation. The process parameters were optimized using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound intensity. It has been observed that ultrasound-assisted lipase-catalyzed hydrolysis of WCO would be a promising alternative for conventional methods. A maximum conversion of 75.19% was obtained at mild operating parameters: molar ratio of oil to water (buffer pH 7) 3:1, catalyst loading of 1.25% (w/w), lower ultrasound power 100 W (ultrasound intensity – 7356.68 W m⁻²), duty cycle 50% and temperature (50 °C) in a relatively short reaction time (2 h). The activation energy and thermodynamic study shows that the hydrolysis reaction is more feasible when ultrasound is combined with mechanical agitation as compared with the ultrasound alone and simple conventional stirring technique. Application of ultrasound considerably reduced the reaction time as compared to conventional reaction. The successive use of the catalyst for repetitive cycles under the optimum experimental conditions resulted in a loss of enzymatic activity and also minimized the product conversion.     

Studies to control biofilm formation by coupling ultrasonication of natural waters and anodization of titanium

The main objective of this study was to investigate the combined effect of ultrasonication of natural waters and anodization of titanium on microbial density and biofilm formation tendency on titanium surfaces. Application of 24 kHz, 400 W high power ultrasound through a 14 mm horn type SS (stainless steel) Sonicator with medium amplitude of 60% for 30 min brought about three order decrease in total bacterial density of laboratory tap water, cooling tower water and reservoir water and two order decrease in seawater. Studies on the effect of ultrasonication on dilute pure cultures of Gram-negative and Gram-positive bacteria showed five order and three order decrease for Pseudomonas sp. and Flavobacterium sp. respectively and two order and less than one order decrease for Bacillus sp. and Micrococcus sp. respectively. Ultrasonication increased lag phase and reduced logarithmic population increase and specific growth rate of Gram-negative bacteria whereas for Gram-positive bacteria specific growth rate increased. Studies on the biofilm formation tendency of these ultrasonicated mediums on titanium surface showed one order reduction under all conditions. Detailed biofilm imaging by advanced microscopic techniques like AFM, SEM and epifluorescence microscopy clearly visualized the lysed/damaged cells and membrane perforations due to ultrasonication. Combination of ultrasonication and anodization brought about maximum decrease in bacterial density and biofilm formation with greater than two order decrease in seawater, two order decrease in Bacillus sp. culture and more than four order decrease in Flavobacterium sp. culture establishing the synergistic effect of anodization and ultrasonication in this study.     

Ultrasound assisted acid catalyzed lactose hydrolysis: Understanding into effect of operating parameters and scale up studies

The current work deals with the value addition of lactose by transforming into hydrolyzed lactose syrup containing glucose and galactose in major proportion using the novel approach of ultrasound assisted acid catalyzed lactose hydrolysis. The hydrolysis of lactose was performed in ultrasonic bath (33 kHz) at 50% duty cycle at different temperatures as 65 °C and 70 °C and two different hydrochloric acid (HCl) concentrations as 2.5 N and 3 N. It was observed that acid concentration, temperature and ultrasonic treatment were the major factors in deciding the time required to achieve ∼90% hydrolysis. The ultrasonic assisted approach resulted in reduction in the reaction time and the extent of intensification was established to be dependent on the temperature, acid concentration and time of ultrasonic exposure. It was observed that the maximum process intensification obtained by introduction of ultrasound in the lactose hydrolysis process performed at 70 °C and 3 N HCl was reduction in the required time for ∼90% hydrolysis from 4 h (without the presence of ultrasound) to 3 h. The scale-up study was also performed using an ultrasonic bath with longitudinal horn (36 kHz as operating frequency) at 50% duty cycle, optimized temperature of 70 °C and acid concentration of 3 N. It was observed that the reaction was faster in the presence of ultrasound and stirring by axial impeller at rpm of 225 ± 25. The time required to complete ∼90% of hydrolysis remained almost the same as observed for small scale study on ultrasonic bath (33 kHz) at 50% duty cycle. The use of recovered lactose from whey samples instead of pure lactose did not result in any significant changes in the progress of hydrolysis, confirming the efficacy of the selected approach. Overall, the work has presented a novel ultrasound assisted approach for intensified lactose hydrolysis.     

Ultrasound assisted enzyme catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate

The present work illustrates the transesterification of glycerol to glycerol carbonate (GlyC) from dimethyl carbonate (DMC) using commercial immobilized lipase (Novozym 435) under ultrasonic irradiation. The experiments were performed in a batch reactor placed in an ultrasonic water bath using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound power on the conversion of glycerol to GlyC. It has been found that ultrasound-assisted lipase-catalyzed transesterification of glycerol would be a potential alternative to conventional alkali-catalyzed method, as high conversion (99.75%) was obtained at mild operating conditions: molar ratio of DMC to glycerol 3:1, catalyst amount of 13% (w/w), lower power input (100 W), duty cycle 50% and temperature (60 °C) in a relatively short reaction time (4 h) using Novozym 435 as catalyst. Ultrasound reduces the reaction time up to 4 h as compared to conventional stirring method (14 h) catalyzed by Novozym 435. The repeated use of the catalyst under the optimum experimental condition resulted in decay in both enzyme activity and product conversion.     

Ultrasound assisted extraction of β-carotene from Spirulina platensis

This paper illustrates the Ultrasound Assisted Extraction (UAE) of β-carotene from Spirulina platensis. Various parameters such as extraction time, solvent type, biomass to solvent ratio, temperature, electrical acoustic intensity, length of the probe tip dipped into the solvent, duty cycle and pre treatment effect were explored for the extraction of β-carotene. From economic point of view, the optimal conditions for the extraction of β-carotene from Spirulina were 1.5 g Spirulina (2 min pre soaked in methanol) in 50 ml n-heptane at 30 °C temperature, 167 W/cm² electrical acoustic intensity and 61.5% duty cycle for 8 min with probe tip length of 0.5 cm dipped into the extracting solvent from the surface. The maximum extraction achieved under the above mentioned optimum parameters was 47.10%. The pre-treatment time showed a promising effect on the yield as pre-treating the biomass with methanol for 2 min before ultrasonication showed 12 times increase in extraction yield of β-carotene.     

Synergism between ultrasonic pretreatment and white rot fungal enzymes on biodegradation of wheat chaff

Lignocellulosic biomass samples (wheat chaff) were pretreated by ultrasound (US) (40 kHz/0.5 W cm⁻²/10 min and 400 kHz/0.5 W cm⁻²/10 min applied sequentially) prior to digestion by enzyme extracts obtained from fermentation of the biomass with white rot fungi (Phanerochaete chrysosporium or Trametes sp.). The accessibility of the cellulosic components in wheat chaff was increased, as demonstrated by the increased concentration of sugars produced by exposure to the ultrasound treatment prior to enzyme addition. Pretreatment with ultrasound increased the concentration of lignin degradation products (guaiacol and syringol) obtained from wheat chaff after enzyme addition. In vitro digestibility of wheat chaff was also enhanced by the ultrasonics pretreatment in combination with treatment with enzyme extracts. Degradation was enhanced with the use of a mixture of the enzyme extracts compared to that for a single enzyme extract.     

Correlation between iodide dosimetry and terephthalic acid dosimetry to evaluate the reactive radical production due to the acoustic cavitation activity

Acoustic cavitation plays an important role in sonochemical processes and the rate of sonochemical reaction is influenced by sonication parameters. There are several methods to evaluate cavitation activity such as chemical dosimetry. In this study, to comparison between iodide dosimetry and terephthalic acid dosimetry, efficacy of sonication parameters in reactive radical production has been considered by iodide and terephthalic acid dosimetries. For this purpose, efficacy of different exposure parameters on cavitations production by 1 MHz ultrasound has been studied. The absorbance of KI dosimeter was measured by spectrophotometer and the fluorescence of terephthalic acid dosimeter was measured using spectrofluorometer after sonication. The result of experiments related to sonication time and intensity showed that with increasing time of sonication or intensity, the absorbance is increased. It has been shown that the absorbance for continuous mode is remarkably higher than for pulsing mode (p-value < 0.05). Also results show that with increasing the duty cycles of pulsed field, the inertial cavitation activity is increased. With compensation of sonication time or intensity in different duty cycles, no significant absorbance difference were observed unless 20% duty cycle. A significant correlation between the absorbance and fluorescence intensities (count) at different intensity (R = 0.971), different sonication time (R = 0.999) and different duty cycle (R = 0.967) were observed (p-value < 0.05). It is concluded that the sonication parameters having important influences on reactive radical production. These results suggest that there is a correlation between iodide dosimetry and terephthalic acid dosimetry to examine the acoustic cavitation activity in ultrasound field.     

Investigation of process variables and intensification effects of ultrasound applied in oxidative desulfurization of model diesel over MoO3/Al2O3 catalyst

A new heterogeneous sonocatalytic system consisting of a MoO₃/Al₂O₃ catalyst and H₂O₂ combined with ultrasonication was studied to improve and accelerate the oxidation of model sulfur compounds of diesel, resulting in a significant enhancement in the process efficiency. The influence of ultrasound on properties, activity and stability of the catalyst was studied in detail by means of GC-FID, PSD, SEM and BET techniques. Above 98% conversion of DBT in model diesel containing 1000 μg/g sulfur was obtained by new ultrasound-assisted desulfurization at H₂O₂/sulfur molar ratio of 3, temperature of 318 K and catalyst dosage of 30 g/L after 30 min reaction, contrary to the 55% conversion obtained during the silent process. This improvement was considerably affected by operation parameters and catalyst properties. The effects of main process variables were investigated using response surface methodology in silent process compared to ultrasonication. Ultrasound provided a good dispersion of catalyst and oxidant by breakage of hydrogen bonding and deagglomeration of them in the oil phase. Deposition of impurities on the catalyst surface caused a quick deactivation in silent experiments resulting only 5% of DBT oxidation after 6 cycles of silent reaction by recycled catalyst. Above 95% of DBT was oxidized after 6 ultrasound-assisted cycles showing a great improvement in stability by cleaning the surface during ultrasonication. A considerable particle size reduction was also observed after 3 h sonication that could provide more dispersion of catalyst in model fuel.     

Green synthesis of nanocomposites consisting of silver and protease alpha chymotrypsin

The synergy of ultrasonication and the exposure to light radiation was found to be necessary in the formation of nanocomposites of silver and a protease alpha chymotrypsin. The reaction was carried out in aqueous medium and the process took just less than 35 min. Ultrasonication alone formed very negligible number of nanoparticles of <100 nm size whereas light alone produced enough number but the size of the particles was >100 nm.The effects of pH (in the range of 3–5, 9–10), ultrasonication time periods (0–30 min), ultrasonication intensity (33–83 W cm^?2), energy of light radiation (short UV, long UV and Fluorescent light) and time period of exposure (5–60 min) to different light radiations were studied.The formation of nanocomposites under these effects was followed by surface plasmon resonance (SPR) spectra, dynamic light scattering (DLS), transmission electron microscopy (TEM). Ag–chymotrypsin nanocomposites of sizes ranging from 13 to 72 nm were formed using the synergy of ultrasonication and exposure to short UV radiation. Results show that ultrasonication promoted nuclei formation, growth and reduction of polydispersity by Ostwald ripening.     

Synergistic effects of combining ultrasound with the Fenton process in the degradation of Reactive Blue 19

The decoloration of reactive dye C.I. Reactive Blue 19 (RB 19) using combined ultrasound with the Fenton process has been investigated. The effect of varying the concentrations of hydrogen peroxide and iron sulfate, initial pH, ultrasonic power, initial dye concentration and dissolved gas on the decoloration and degradation efficiencies was measured. Calibration of the ultrasound systems was performed using calorimetric measurements and oxidative species monitoring using the Fricke dosimeter and degradations were carried out with a 20 kHz probe type transducer at 2, 4, 6 and 8 W cm⁻² of acoustic intensity at 15, 25, 50 and 75 mg L⁻¹ initial dye concentrations. First order rate kinetics was observed. It was found that while the degradation rate due to ultrasound alone was slow, sonication significantly accelerated the Fenton reaction. While the results were similar to those reported for other dyes, the effects occurred at lower concentrations. The rate and extent of decoloration of RB 19 increased with rising hydrogen peroxide concentration, ultrasonic powers and iron sulfate concentration but decreased with increasing dye concentration. An optimum pH value of pH = 3.5 was found. The rate of decoloration was higher when dissolved oxygen was present as compared with nitrogen and argon confirming the solution phase mechanism of the degradation.     

Effect of ultrasound on the extraction of total anthocyanins from Purple Majesty potato

This study examined anthocyanin extraction using the application of ultrasound to raw freeze dried, microwaved and raw sliced Purple Majesty potato, a new pigmented potato variety rich in anthocyanins. A 20 kHz probe was used for the sonication at 3 different amplitudes (30%, 50% and 70%) and ethanol in water at different ratios (50:50 and 70:30 v/v) was used for the extraction. Anthocyanin extraction from raw freeze dried purple potato was optimal at an ethanol:water ratio (70:30; v/v) after 5 min of ultrasonication, while the least amount of anthocyanins was extracted from raw sliced potatoes. The application of microwaves (as a pre-treatment) before the UAE resulted in an increase in the amount of anthocyanins extracted and a decrease in the amount of solvent used. Analysis of variance showed that potato form, ultrasonication time, ultrasonication amplitude and solvent ratio as well as two and three way interactions between some of these factors had a very significant effect (p < 0.000) on the amount of anthocyanins extracted.     

Ultrasound-assisted mineralization of organic contaminants using a recyclable LaFeO3 and Fe3+/persulfate Fenton-like system

A recyclable heterogeneous catalyst has been successfully developed for application in a Fenton-type advanced oxidation process without adding external H₂O₂. LaFeO₃ was prepared from Fe(NO₃)₃·9H₂O and La(NO₃)·6H₂O by a simple sol-gel method and its catalytic efficiency was evaluated for mineralization of 4-chlorophenol using a Fenton-like process. The mineralization process was carried out under ultrasonication in presence of heterogeneous LaFeO₃ catalyst with H₂O₂ that was produced during ultrasonication. The mineralization process was monitored through total organic carbon (TOC) analysis. Very importantly, utmost 5-fold synergism was evidenced by the ultrasound mediated LaFeO₃-catalyzed system. Besides, more than twofold synergism was observed by combining the ultrasound assisted LaFeO₃ catalytic process and potassium persulfate (KPS) assisted advanced oxidation process. It is worth to mention that complete mineralization (∼96%) of 4-chlorophenol (initial concentration of 1.25 × 10⁻⁴ M) was observed within 1 h in the presence of LaFeO₃ (0.5 g L⁻¹) and KPS (1.0 mmol) under ultrasonication (40 kHz). Even after four cycles, the activity of LaFeO₃ remained intact which proved its recyclability. Extremely reusable heterogeneous LaFeO₃ catalyst makes the system more interesting from both economic and environmental points of view.