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.     

Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein

The effects of ultrasonic frequency mode, power density, pretreatment time and other parameters under low power density on the degree of hydrolysis (DH) of defatted wheat germ protein (DWGP) and angiotensin-I-converting enzyme (ACE) inhibitory activity of DWGP hydrolysate were studied in this research. Ultraviolet–visible (UV–Vis) spectra, free sulfhydryl (SH), disulfide bond (SS), surface hydrophobicity and hydrophobic protein content of ultrasound-pretreated protein and hydrophobic amino acid (HAA) content of alcalase-hydrolysate of DWGP were measured under optimized ultrasonic condition. The ultrasonic frequency mode with dual-fixed frequency combination of 28/40 kHz showed higher ACE inhibitory activity of DWGP hydrolysate compared with that of other ultrasound frequency modes and all the ultrasonic frequency combinations involving in 28 kHz showed higher ACE inhibitory activity. Under the dual-fixed frequency ultrasound mode of 28/40 kHz, ultrasonic power density of 60 W/L, pretreatment time of 70 min, temperature of 60°C and substrate concentration of 60 g/L, the ACE inhibitory activity of DWGP hydrolysate was the highest with its value of 74.75% (increased by 62.30% compared to control). However, all the ultrasonic pretreatment did not increase the DH of DWGP significantly (p > 0.05). The changes in UV–Vis spectra, SH and SS groups, surface hydrophobicity and hydrophobic protein content indicated that the structure of DWGP unfolded after ultrasound pretreatment. The HAA content of hydrolysate from the pretreated DWGP increased significantly (p < 0.05). The results proved that ultrasound pretreatment loosed the protein structure and exposed more HAA residues of protein to be attacked easily by alcalase. This resulted in the increase in the HAA content which related to the ACE inhibitory activity.     

Sonophotocatalytic inactivation of E. coli using ZnO nanofluids and its mechanism

The present study evaluated inactivation efficiency of a sonophotocatalytic process using ZnO nanofluids including ultrasonic parameters such as power density, frequency and time. The result showed that inactivation efficiency was increased by 20% when ultrasonic irradiation was combined with photocatalytic process in the presence of natural light. Comparison of inactivation efficiency in photocatalytic, ultrasonic and sonocatalytic processes using Escherichia coli as a model bacteria identified that inactivation efficiencies are shown in the following order: ultrasonic irradiation < sonocatalysis < photocatalysis < sonophotocatalysis. Furthermore, inactivation mechanism of sonophotocatalysis was proposed. Studies of reactive oxygen species (ROS) and zinc ions (Zn²⁺) release evaluation revealed that ROS play a key role in bacterial inactivation rather than Zn²⁺. Permeability of outer membrane (OM) and inner membrane (IM) of E. coli bacterial cells were studied and exhibited that sonophotocatalysis increased the permeability of OM and IM significantly. The enhanced bacterial inactivation effect in sonophotocatalytic process contributed to acoustic cavitation, sonocatalysis of ZnO and sonoporation phenomenon.     

Intensification of extraction of curcumin from Curcuma amada using ultrasound assisted approach: Effect of different operating parameters

Curcumin, a dietary phytochemical, has been extracted from rhizomes of Curcuma amada using ultrasound assisted extraction (UAE) and the results compared with the conventional extraction approach to establish the process intensification benefits. The effect of operating parameters such as type of solvent, extraction time, extraction temperature, solid to solvent ratio, particle size and ultrasonic power on the extraction yield have been investigated in details for the approach UAE. The maximum extraction yield as 72% was obtained in 1 h under optimized conditions of 35 °C temperature, solid to solvent ratio of 1:25, particle size of 0.09 mm, ultrasonic power of 250 W and ultrasound frequency of 22 kHz with ethanol as the solvent. The obtained yield was significantly higher as compared to the batch extraction where only about 62% yield was achieved in 8 h of treatment. Peleg’s model was used to describe the kinetics of UAE and the model showed a good agreement with the experimental results. Overall, ultrasound has been established to be a green process for extraction of curcumin with benefits of reduction in time as compared to batch extraction and the operating temperature as compared to Soxhlet extraction.     

Fermentation of Saccharomyces cerevisiae in a 7.5 L ultrasound-enhanced fermenter: Effect of sonication conditions on ethanol production,intracellular Ca2+ concentration and key regulating enzyme activity in glycolysis

In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca²⁺ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.     

Mapping of an ultrasonic bath for ultrasound assisted extraction of mangiferin from Mangifera indica leaves

The present work deals with the mapping of an ultrasonic bath for the maximum extraction of mangiferin from Mangifera indica leaves. I₃⁻ liberation experiments (chemical transformations) and extraction (physical transformations) were carried out at different locations in an ultrasonic bath and compared. The experimental findings indicated a similar trend in variation in an ultrasonic bath by both these methods. Various parameters such as position and depth of vessel in an ultrasonic bath, diameter and shape of a vessel, frequency and input power which affect the extraction yield have been studied in detail. Maximum yield of mangiferin obtained was approximately 31 mg/g at optimized parameters: distance of 2.54 cm above the bottom of the bath, 7 cm diameter of vessel, flat bottom vessel, 6.35 cm liquid height, 122 W input power and 25 kHz frequency. The present work indicates that the position and depth of vessel in an ultrasonic bath, diameter and shape of a vessel, frequency and input power have significant effect on the extraction yield. This work can be used as a base for all ultrasonic baths to obtain maximum efficiency for ultrasound assisted extraction.     

Effect of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system

The objective of this study was to investigate the efficacy of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system. Ultrasound power level (0–68.5 W) and sonication time (0–9 min) at 20 °C were assessed against the growth of L. sakei using a Microplate reader over a period of 24 h. The L. sakei growth data showed a good fit with the Gompertz model (R² > 0.90; SE < 0.042). Second order polynomial models demonstrated the effect of ultrasonic power and sonication time on the specific growth rate (SGR, μ, h⁻¹) and lag phase (λ, h). A higher SGR and a shorter lag phase were observed at low power (2.99 W for 5 min) compared to control. Conversely, a decrease (p < 0.05) in SGR with an increase in lag phase was observed with an increase in ultrasonic power level. Cell-free extracts obtained after 24 h fermentation of ultrasound treated samples showed antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium at lower concentrations compared to control. No significant difference (p < 0.05) among treatments was observed for lactic acid content after a 24 h fermentation period. This study showed that both stimulation and retardation of L. sakei is possible, depending on the ultrasonic power and sonication time employed. Hence, fermentation process involving probiotics to develop functional food products can be tailored by selection of ultrasound processing parameters.     

Effects of ultrasonic agitation and surfactant additive on surface roughness of Si (1 1 1) crystal plane in alkaline KOH solution

In the silicon wet etching process, the “pseudo-mask” formed by the hydrogen bubbles generated during the etching process is the reason causing high surface roughness and poor surface quality. Based upon the ultrasonic mechanical effect and wettability enhanced by isopropyl alcohol (IPA), ultrasonic agitation and IPA were used to improve surface quality of Si (1 1 1) crystal plane during silicon wet etching process. The surface roughness R_q is smaller than 15 nm when using ultrasonic agitation and R_q is smaller than 7 nm when using IPA. When the range of IPA concentration (mass fraction, wt%) is 5–20%, the ultrasonic frequency is 100 kHz and the ultrasound intensity is 30–50 W/L, the surface roughness R_q is smaller than 2 nm when combining ultrasonic agitation and IPA. The surface roughness R_q is equal to 1 nm when the mass fraction of IPA, ultrasound intensity and the ultrasonic frequency is 20%, 50 W and 100 kHz respectively. The experimental results indicated that the combination of ultrasonic agitation and IPA could obtain a lower surface roughness of Si (1 1 1) crystal plane in silicon wet etching process.     

Variation of dissolved organic nitrogen concentration during the ultrasonic pretreatment to Microcystis aeruginosa

Algae cells were the main sources of dissolved organic nitrogen (DON) in raw water with plenty of algae, and ultrasonic pretreatment was one of the algae-controlling methods through the damage of algae cells. However, the variation of DON concentration during the ultrasonic treatment process was not confirmed. Variation of DON concentration during the processes of low frequency ultrasound treatment of Microcystis aeruginosa was investigated. In addition, the effect of sonication on the metabolite concentration, algae cellar activity and the subsequent coagulation performance were discussed. The results showed that after a long duration of ultrasonic (60 s), nearly 90% of the algal cells were damaged and the maximum concentration of DON attained more than 3 mg/L. In order to control the leakage extent of DON, the sonication time should be less than 30 s with power intensity of more than 1.0 W/cm³. In the mean time, ultrasonic treatment could inhibit the reactivation and the proliferation of algal, keep the algae cell wall integrity and enhance coagulation effectively under the same condition. However, ultrasound frequency had little effect on DON at the frequency range used in this study (20–150 kHz).     

Experimental study on the influence of low-frequency and low-intensity ultrasound on the permeability of the Mycobacterium smegmatis cytoderm and potentiation with levofloxacin

Tuberculosis is an infectious disease caused by the bacterium M. tuberculosis. The aim of this study was to investigate the bactericidal effect and underlying mechanisms of low-frequency and low-intensity ultrasound combined with levofloxacin treatment against M. smegmatis (a surrogate of M. tuberculosis). As part of this study, M. smegmatis was continuously irradiated with low frequency ultrasound (42 kHz) using several different doses whereby both intensity (0.138, 0.190 and 0.329 W/cm²) and exposure time (5, 15 and 20 min) were varied. Flow cytometric analyses revealed that the permeability of M. smegmatis increased following ultrasound exposure. The survival rate, structure and morphology of bacteria in the lower-dose (I_SATA = 0.138 W/cm² for 5 min) ultrasound group displayed no significant differences upon comparison with the untreated group. However, the survival rate of bacteria was significantly reduced and the bacterial structure was damaged in the higher-dose (I_SATA = 0.329 W/cm² for 20 min) ultrasound group. Ultrasound irradiation (0.138 W/cm²) was subsequently applied to M. smegmatis in combination with levofloxacin treatment for 5 min. The results demonstrated that the bactericidal effect of ultrasonic irradiation combined with levofloxacin is higher compared to ultrasound alone or levofloxacin alone.     

Ultrasound-intensified laccase production from Trametes versicolor

An efficient intermittent ultrasonic treatment strategy was developed to improve laccase production from Trametes versicolor mycelia cultures. The optimized strategy consisted of exposing 2-day-old mycelia cultures to 5-min ultrasonic treatments for two times with a 12-h interval at the fixed ultrasonic power and frequency (120 W, 40 kHz). After 5 days of culture, this strategy produced the highest extracellular laccase activity of 588.9 U/L among all treatments tested which was 1.8-fold greater than the control without ultrasound treatment. The ultrasonic treatment resulted in a higher pellet porosity that facilitated the mass transfer of nutrients and metabolites from the pellets to the surrounding liquid. Furthermore, the ultrasonic treatment induced the expression of the laccase gene (lcc), which correlated with a sharp increase in both extracellular and intracellular laccase activity. This is the first study to find positive effects of ultrasound on gene expression in fungal cells. These results provide a basis for understanding the stimulation of metabolite production and process intensification by ultrasonic treatment in filamentous fungal culture.     

Inactivation of Escherichia coli and Streptococcus mutans by ultrasound at 500 kHz

This paper shows a systematic study of the 500 kHz frequency ultrasound efficiency on the microbial inactivation as a function of ultrasonic power delivered into the bacterial suspension. The inactivation of Escherichia coli IAM 12058, a Gram-negative bacterium and Streptococcus mutans JCM 5175, a Gram-positive bacterium is enhanced by increasing the ultrasonic power in the range of 1.7–12.4 W and the logarithm of survival ratio decreases linearly with irradiation time, except for E. coli sonicated with the highest power level. The rate constants were estimated in the linear region of the plots representing survival ratio logarithm vs. sonication time. A better understanding of the inactivation process at 500 kHz could be gained by suppressing the chemical effects with a radical scavenger. We find out that the rate constants increase with the ultrasonic power delivered into the solution and dramatically decrease by the addition of t-butanol as a radical scavenger to the bacterial suspension. For comparison, experiments were carried out at a low frequency level of 20 kHz. It was found out that for the same ultrasonic power delivered into the bacterial suspension, the inactivation was slightly enhanced at 500 kHz frequency. The examinations of bacterium performed with a TEM revealed lethal damages arising from the interaction of bacterial cells with the cavitational bubbles. A significant amount of empty cell envelopes as well as their cytoplasmatic content was detected. Thus, based on these new data, the mechanism of bacterial inactivation by ultrasounds at high frequency is discussed here.     

Ultrasound-assisted transesterification of soybean oil using low power and high frequency and no external heating source

In this work, high frequency and low power ultrasound without external heating source and mechanical stirring in biodiesel production were studied. Transesterification of soybean oil with methanol and catalyzed by KOH was investigated using ultrasound equipment and ultrasonic transducer. The effect of ultrasonic output power (3 W–9 W), ultrasonic frequency (1 MHz and 3 MHz), and alcohol to oil molar ratio (6:1 and 8:1) have been investigated. The increase in ultrasonic power provided higher conversion rates. In addition, higher conversion rates were obtained by increasing the ultrasonic frequency from 1 MHz to 3 MHz (48.7% to 79.5%) for the same reaction time. Results also indicate that the speed of sound can be used to evaluate the produced biodiesel qualitatively. Further, the ultrasound system presented electric consumption (46.2 W∙h) four times lower than achieved using the conventional method (211.7 W∙h and 212.3 W∙h). Thus, biodiesel production using low power ultrasound in the MHz frequency range is a promising technology that could contribute to biodiesel production processes.     

Ultrasonic preparation of cationic cotton and its application in ultrasonic natural dyeing

Cationization of cotton fabric was conferred by the sonicator reaction of cellulose with bromoacetyl bromide, followed by substitution of the terminal bromo groups by triethylamine. Experiments showed that the optimal volume of bromoacetyl bromide necessary to succeed the first stage was 0.4 mL. The order of weight gain for various processes indicates, ultrasound, 25 kHz > ultrasound, 40 kHz > mechanical stirring. Also, for the second stage the order of nitrogen contents indicates ultrasound, 25 kHz > ultrasound, 40 kHz > mechanical stirring. The structures of both untreated and cationic fibres were investigated by FTIR spectroscopy. Modified cotton fabric was subsequently dyed in both conventional and ultrasonic techniques with isosalipurposide dye isolated from Acacia cyanophylla yellow flowers. The effect of dye bath pH, ultrasonic power and frequency, dyeing time and temperature were studied and the order of K/S values indicates ultrasound, 25 kHz > ultrasound, 40 kHz > CH. ultrasound was also found to enhance the dye uptake and the overall fastness properties. Analysis of the sorption isotherms of isosalipurposide dye on cationic cotton fabric shows that the Languimir isotherm equation is best able to correlate the data.     

Optimization of ultrasound-assisted extraction of bioactive compounds from wild garlic (Allium ursinum L.)

Ultrasound-assisted extraction was used for extraction of bioactive compounds and for production of Allium ursinum liquid extract. The experiments were carried out according to tree level, four variables, face-centered cubic experimental design (FDC) combined with response surface methodology (RSM). Temperature (from 40 to 80 °C), ethanol concentration (from 30% to 70%), extraction time (from 40 to 80 min) and ultrasonic power (from 19.2 to 38.4 W/L) were investigated as independent variables in order to obtain the optimal conditions for extraction and to maximize the yield of total phenols (TP), flavonoids (TF) and antioxidant activity of obtained extracts. Experimental results were fitted to the second order polynomial model where multiple regression and analysis of variance were used to determine the fitness of the model and optimal condition for investigated responses. The predicted values of the TP (1.60 g GAE/100 g DW), TF (0.35 g CE/100 g DW), antioxidant activity, IC₅₀ (0.71 mg/ml) and EY (38.1%) were determined at the optimal conditions for ultrasound assisted extraction: 80 °C temperature, 70% ethanol, 79.8 min and 20.06 W/L ultrasonic power. The predicted results matched well with the experimental results obtained using optimal extraction conditions which validated the RSM model with a good correlation.     

Alternate pulses of ultrasound and electricity enhanced electrochemical process for p-nitrophenol degradation

A novel alternated ultrasonic and electric pulse enhanced electrochemical process was developed and used for investigating its effectiveness on the degradation of p-nitrophenol (PNP) in an aqueous solution. The impacts of pulse mode, pH, cell voltage, supporting electrolyte concentration, ultrasonic power and the initial concentration of PNP on the performance of PNP degradation were evaluated. Possible pathway of PNP degradation in this system was proposed based on the intermediates identified by GC–MS. Experimental results showed that 94.1% of PNP could be removed at 2 h in the dual-pulse ultrasound enhanced electrochemical (dual-pulse US-EC) process at mild operating conditions (i.e., pulse mode of electrochemical pulse time (T_EC) = 50 ms and ultrasonic pulse time (T_US) = 100 ms, initial pH of 3.0, cell voltage of 10 V, Na₂SO₄ concentration of 0.05 M, ultrasonic powder of 48.8 W and initial concentration of PNP of 100 mg/L), compared with 89.0%, 58.9%, 2.4% in simultaneous ultrasound enhanced electrochemical (US-EC) process, pulsed electrochemical (EC) process and pulsed ultrasound (US), respectively. Moreover, energy used in the dual-pulse US-EC process was reduced by 50.4% as compared to the US-EC process. The degradation of PNP in the pulsed EC process, US-EC process and dual-pulse process followed pseudo-first-order kinetics. Therefore, the dual-pulse US-EC process was found to be a more effective technique for the degradation of PNP and would have a promising application in wastewater treatment.     

Ultrasound pretreatment of wheat dried distiller’s grain (DDG) for extraction of phenolic compounds

Wheat Dried distiller’s grain (DDG), a coproduct from the ethanol production process, is rich in potentially health-promoting phenolic compounds. In the extraction of phenolic compounds from DDG, the DDG cell wall is an important barrier for mass transfer from the inside to the outside of the cell. The effect of high-power ultrasound pretreatment on destruction of DDG cell walls and extraction yield and rate was investigated. Direct sonication by an ultrasound probe horn at 24 kHz was applied and factors such as ultrasound power and treatment time were investigated. The method of nitrogen (N₂) adsorption at 77 K was used as a means to determine and compare the changes in physical properties (specific surface area, pore volume and pore size) of the treated samples at different levels of ultrasound power and treatment time. Increasing specific surface area, pore volume and pore size caused by ultrasonic treatment implied development of new or larger pores and damaged cell walls. Also, it was observed that the ultrasound pretreatment of DDG particles increased the extraction yield and rate of phenolic compounds from DDG by 14.29%. Among tested ultrasound conditions, 100% ultrasound power for 30 s was evaluated as the best pretreatment condition.     

Growth and size control in anti-solvent crystallization of glycine with high frequency ultrasound

Antisolvent crystallization of glycine was performed under ultrasonic irradiation of 1.6 MHz. The irradiation enhanced both the growth of α-glycine crystal and the uniformity in the crystal size. The degree of both enhancement effects increased with increasing ultrasonic power. While under the irradiation of 20 kHz ultrasound, no growth enhancement was observed, but the crystal size reduced as was reported in the literature. To elucidate the mechanism of growth enhancement, another experiment was designed and conducted to avoid the effect of nucleation from the sonocrystallization. The result suggests that the ultrasound enhances the incorporation of microcrystals to larger crystals. Probably, the collision between solid particles is intensified by the disturbance characterized by the high frequency ultrasound. The crystal growth was modeled with an apparent reaction of microcrystal and larger crystal. The result of the growth experiment was successfully predicted with a rate equation for pseudo first order reaction with a single parameter of rate constant. The rate constant linearly increased with the ultrasonic power. The analysis enables quantitative evaluation of the ultrasonic effect on the crystal growth.     

Influence of high power ultrasound on Brettanomyces and lactic acid bacteria in wine in continuous flow treatment

The purpose of this study was to investigate the effect of non-thermal technology, a high power ultrasound (HPU) in continuous flow treatment on the reduction in number of Brettanomyces yeasts and lactic acid bacteria (LAB) in wine samples. Yeast cells and lactic acid bacteria were screened for their sensitivity to a high power ultrasound treatment using an ultrasonic processor (400 W, 24 kHz, 100 μm amplitude) at two different wine temperatures (30 and 40 °C). High power ultrasound treatment in continuous flow showed satisfactory reduction of Brettanomyces yeasts (89.1–99.7%) and lactic acid bacteria (71.8–99.3%). Sensory properties of wine were impaired. The results indicate a great potential for the application of HPU in continuous flow system for wine processing in terms of lower usage of SO₂ and preservatives. More attention is needed to preserve sensorial properties of wine.     

Medium-high frequency ultrasound and ozone based advanced oxidation for amoxicillin removal in water

In this study, treatment of an antibiotic compound amoxicillin by medium-high frequency ultrasonic irradiation and/or ozonation has been studied. Ultrasonic irradiation process was carried out in a batch reactor for aqueous amoxicillin solutions at three different frequencies (575, 861 and 1141 kHz). The applied ultrasonic power was 75 W and the diffused power was calculated as 14.6 W/L. The highest removal was achieved at 575 kHz ultrasonic frequency (>99%) with the highest pseudo first order reaction rate constant 0.04 min⁻¹ at pH 10 but the mineralization achieved was around 10%. Presence of alkalinity and humic acid species had negative effect on the removal efficiency (50% decrease). To improve the poor outcomes, ozonation had been applied with or without ultrasound. Ozone removed the amoxicillin at a rate 50 times faster than ultrasound. Moreover, due to the synergistic effect, coupling of ozone and ultrasound gave rise to rate constant of 2.5 min⁻¹ (625 times higher than ultrasound). In the processes where ozone was used, humic acid did not show any significant effect because the rate constant was so high that ozone has easily overcome the scavenging effects of natural water constituents. Furthermore, the intermediate compounds, after the incomplete oxidation mechanisms, has been analyzed to reveal the possible degradation pathways of amoxicillin through ultrasonic irradiation and ozonation applications. The outcomes of the intermediate compounds experiments and the toxicity was investigated to give a clear explanation about the safety of the resulting solution. The relevance of all the results concluded that hybrid advanced oxidation system was the best option for amoxicillin removal.