Eadaoin M. Joyce

The development and evaluation of ultrasound for the treatment of bacterial suspensions. A study of frequency, power and sonication time on cultured Bacillus species☆

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.

Potential uses of ultrasound in the biological decontamination of water.

In the past there was a prevailing feeling in industry that power ultrasound would be too expensive to use for water treatment on an industrial scale. This was based on calculations involving the direct scale up of power consumption in small-scale (generally batch) laboratory experiments. In recent times this attitude has changed somewhat as a result of the installation of a number of ultrasonic devices in operational water or sewage treatment plants. In our laboratories we have investigated the decontamination of water under the influence of ultrasound alone and in conjunction with other treatments. The results, particularly when applied to flowing systems, indicate a real future for sonochemistry in water treatment.

Evaluation of the mechanisms of the effect of ultrasound on Microcystis aeruginosa at different ultrasonic frequencies

Blooms of cyanobacteria are now considered to be a common environmental issue. They are hazardous to both domestic and wild animals and humans. Current treatments are unable to effectively control such blooms as they become tolerant to biocides and it is difficult to degrade cyanobacterial toxins in water. Alternative methods for control are currently under investigation. One potential effective method is ultrasonic irradiation. Ultrasound inactivates algal and cyanobacteria cells through cavitation by generating extreme conditions, resulting in a number of physical, mechanical and chemical effects. The aim of this study was to investigate the effect of ultrasound at different frequencies on Microcystis aeruginosa. Flow cytometry was used to measure cyanobacterial metabolic cell viability in addition to the more commonly used haemocytometry, optical density and fluorimetry. Results indicate low frequency 20 kHz ultrasound with high intensity (0.0403 W cm(-3)) is effective for the inactivation of cyanobacterial cells. Higher frequencies of 580 kHz (0.0041 W cm(-3)) also resulted in an inactivation effect, but 1146 kHz (0.0018 W cm(-3)) showed a declumping effect as evidenced by flow cytometry. Ultrasonic treatment over time under different sonication conditions demonstrates the following: 1. Acoustic cavitation via mechanical effects can induce sufficient shear forces to directly rupture cyanobacteria cells. 2. At higher ultrasonic frequencies the mechanical energy of cavitation is less but a larger proportion of free radicals are produced from the ultrasonic degradation of water, which chemically attacks and weakens the cyanobacteria cell walls. 3. At higher frequencies free radicals also damage chlorophyll a leading to a loss in photosynthetic cell viability. 4. At low powers ultrasonic energy results in declumping of cyanobacteria.

Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry

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).

The development and evaluation of electrolysis in conjunction with power ultrasound for the disinfection of bacterial suspensions

There is an increasing incidence in health problems related to environmental issues that originate from inadequate treatment of potable waters. This has compelled scientists and engineers to engage in innovative technologies to achieve a maximum disinfection at affordable costs. Some species of bacteria produce colonies and spores that can agglomerate in spherical clusters and thus protect organisms on the inside of the cluster against biocidal attack. Flocs of fine particles (e.g., clay) can entrap bacteria and this can also protect them against the biocides. Other bacteria have the ability to mutate, thus building up resistance to conventional biocides (e.g., chlorine). Ultrasound has been shown to be effective in improving the effectiveness of biocides such as chlorine. The aim of this present study was to investigate the effect of electrolysis and power ultrasound as a disinfection treatment and to provide a greater knowledge of the fundamentals of disinfection through the production of hypochlorite in situ from saline solution via electrolysis. The electrode materials investigated were, carbon (felt and graphite), copper and stainless steel rods. The results show that sonication appears to amplify the effect of electrolysis. A combination of both treatments is significantly better than sonication or electrolysis alone.

Effect of ultrasonic frequency and power on algae suspensions

Cyanobacteria are photosynthetic bacteria with some characteristics of algae. Some cyanobacteria produce toxins that have been shown to be hazardous to both animals and humans. Previous research has demonstrated power ultrasound can provide a suitable method to control algae blooms although the optimum ultrasonic parameter settings have not been determined to ensure an effective and energy efficient treatment. In this work the effect of ultrasound on suspensions of Microcystis aeruginosa has been investigated at the following frequencies 20, 40, 580, 864 and 1146 kHz. Results showed that the reduction in algal numbers is dependent on both frequency and intensity. In order to quantify the effect we have defined the efficiency of the ultrasonic control of algae at a specific frequency as: (% inactivation of the algae) / (ultrasonic intensity applied). When this is applied to the results at different frequencies the order of efficiency for algae reduction is 20 < 1146 < 864 < 580 kHz. This suggests that ultrasound can offer a suitable method for algae inactivation or control but the sonication conditions must be taken into account.

Effect of ultrasonic frequency and power on the disruption of algal cells.

In this work the effect of ultrasonic waves on suspensions of Chlamydomonas concordia and Dunaliella salina have been investigated at frequencies of 20, 585, 864 and 1146 kHz and at different acoustic powers. Results showed that the reduction in algal numbers was dependent on both frequency and acoustic power. The order of efficiency of the ultrasonic disruption of C. concordia at different frequencies was 20 < 580 < 864 < 1146 kHz, and for D. salina was 20< 580 ≅ 864 ⩽ 1146 kHz. It is clear that high-frequency sonication is more effective than conventional low-frequency sonication for the disruption of cells for both species. Results showed that suitable disruption frequencies for each algae were associated with the mechanical properties of the cell. The frequency dependence of the efficiency of algae disruption on the mechanical resonances of both the algae cell is discussed in terms of bubble oscillation in an ultrasonic field.

Enzymatic pre-treatment as a means of enhancing the antibacterial activity and stability of ZnO nanoparticles sonochemically coated on cotton fabrics

Zinc oxide nanoparticles (ZnO NPs) are known for their excellent antibacterial properties. This paper describes a method for enhancing the stability and the antibacterial activity of ZnO NPs synthesized and embedded sonochemically on cotton fabrics, by pre-treating the fabric surface with cellulase enzyme. The enzymatic pre-treatment resulted in the deposition of smaller sized NPs with improved adhesion. The reduction in particle size brought about better antibacterial performance against several types of bacteria. The sonochemically produced ZnO coating withstood 10 laundry cycles at 92 °C retaining its antibacterial activity.

Effect of sonication frequency on the disruption of algae.

In this study, the efficiency of ultrasonic disruption of Chaetoceros gracilis, Chaetoceros calcitrans, and Nannochloropsis sp. was investigated by applying ultrasonic waves of 0.02, 0.4, 1.0, 2.2, 3.3, and 4.3 MHz to algal suspensions. The results showed that reduction in the number of algae was frequency dependent and that the highest efficiency was achieved at 2.2, 3.3, and 4.3MHz for C. gracilis, C. calcitrans, and Nannochloropsis sp., respectively. A review of the literature suggested that cavitation, rather than direct effects of ultrasonication, are required for ultrasonic algae disruption, and that chemical effects are likely not the main mechanism for algal cell disruption. The mechanical resonance frequencies estimated by a shell model, taking into account elastic properties, demonstrated that suitable disruption frequencies for each alga were associated with the cells mechanical properties. Taken together, we consider here that physical effects of ultrasonication were responsible for algae disruption.

Combined Effect of Ultrasound and Ozone on Bacteria in Water

The aim of this study is to assess the synergetic effect of combined ultrasound and ozone treatment on the biological disinfection of water on a large-scale application using viable plate counts and flow cytometry. Escherichia coli B bacteria in saline suspension was treated using a commercially available combined ultrasound and ozone system (USO3 (Ultrasonic Systems Gmbh)) for 16 min. Two analytical methods were used to assess the results in terms of live and dead cells in the bulk liquid: standard viable plate counting recorded in terms of colony forming units per milliliter and flow cytometry. In the latter case 1 mL of bacterial suspension was stained simultaneously with the fluorescent stains SYTO9 and propidium iodide (PI). Transmission electron microscopy was used to generate images identifying the biological effects of different treatments using ultrasound and ozone on bacterial cell walls. Results demonstrated that treatment with ozone alone (1 mg/L) resulted in a significant reduction (93%) in the number of live cells after 16 min treatment whereas ultrasound alone showed only a small reduction (24%). However, a combination of ozone and ultrasound showed a synergistic effect and enhanced the inactivation to 99% after 4 min. A combined ultrasound and ozone treatment of bacterial suspensions using a commercial system affords a promising method for water disinfection that is better than treatment using either method alone. Standard viable plate count analysis is normally used to assess the effectiveness of disinfection treatments; however flow cytometry proved to be a more sensitive method to determine the actual effects in terms of not only live and dead cells but also damaged cells. This type of analysis (cell damage) is difficult if not impossible to achieve using traditional plate counting methodology.