0.001 0.01 0.1 1 10 100 0 5 10 15 20 25 Time (mins) % 850kHz 1.3W 850kHz 19.31W 20kHz 21.4W 20kHz 3.18W 0.001 0.01 0.1 1 10 100 1000 -2 0 2 4 6 8 10 12 14 16 Time (mins) % 850kHz 1.3W 850kHz 19.31W 20kHz 21.4W 20kHz 3.18W THE EFFECT OF SONICATION AT DIFFERENT FREQUENCIES ON MICROBIAL DISINFECTION H.Duckhouse, T.J.Mason, S.S.Phull, L.Paniwnyk and J.P.Lorimer Sonochemistry Centre, School of the S.E., Coventry University, Coventry, CV1 5FB, UK COST D32 Working Group High-Energy Micro-Environments in Biotechnology Abstract Ultrasound can be used to improve the efficiency of a biocide. Previous studies have shown a dramatic frequency effect, depending on the timing of the sonication with respect to biocidal treatment i.e. whether it is sequential or simultaneous. In this study we have investigated the effect of ultrasonic power on the inactivation of suspensions of E. coli using two different frequencies and pre and simultaneous treatment with sodium hyperchlorite. Power does affect the kill rate but the frequency appears to be the major factor in terms of the timing of the ultrasonic treatment. Introduction From the point of view of simplicity and effectiveness chlorination has been used in the past as the biocidal treatment of choice for water [1]. The effectiveness of bacteria kill can be improved by increasing the concentration of chlorine but this may exacerbate some problems associated with chlorination including (a) chemical reactions to produce harmful by-products, (b) the build-up of bacteria resistance, (c) the production of unpleasant flavours and (d) ineffective killing of micro-organisms on the inside of agglomerates. Two solutions to these problems exist: the use of an alternative biocide or a reduction in chlorine concentration but with the same effectiveness e.g. by the use of ultrasound. Ultrasound can improve chlorination through the dispersal of bacterial clumps and by the temporary weakening of cell walls making them more permeable [2,3]. We have reported the influence of ultrasound at two different frequencies and powers before (pre-treatment) and during (simultaneous treatment) chlorination on the destruction of Escherchia coli suspensions, and reported what appears to be a dramatic frequency effect [3]. For the lower frequency of 20kHz (at 21.4W) the improvement in biocidal activity is greatest when the ultrasound is applied at the same time as chlorination. However using the higher frequency of 850kHz (at 1.3W) the improvement is best when ultrasound is used as a pre-treatment immediately followed by hypochlorite addition. The kill rate achieved for pre-treatment using 850kHz and simultaneous treatment using 20kHz are very similar. We have continued our investigations in an attempt to elucidate the mechanism of the frequency effect and in particular whether the difference in the acoustic power employed played a significant role. Method and Results A suspension of Escherchia coli was prepared by an overnight growth at 37°C in a shaking incubator in nutrient broth. This bacterial suspension was centrifuged and washed in saline solution (0.9% NaCl) and were set to a standard turbidity (O.D of 0.17 at 440nm) in a medium of saline solution, equivalent to 1*10 8 bacteria/cm 3 . Serial dilution and standard plate counts were used as the method of analysis. Results are reported as percentage of surviving Colony Forming Units per millilitre (CFUcm -3 ). Sample volumes of suspension (200cm 3 ) were subject to different chlorination treatments. Sonication experiments at 20kHz; 21.4W and 3W (Sonics and Materials VC-600) and 850kHz; 1.3W and 19.31W (Meinhart Ultrachalltechnik K80-5) were performed as follows: (a) Pre-treatment with ultrasound was applied for 1 minute. Immediately after pre-treatment the ultrasound was turned off and a sample was taken for analysis (t = 0). Hypochlorite was added at this point and samples were withdrawn at intervals (t = 1, 2, 5, 10, and 15 minutes). The sonication period prior to chlorination is represented by negative times. (b) Simultaneous treatment ultrasound was applied for 1 minute. After this period silent conditions were observed for the rest of the chlorination period. Samples were taken for analysis immediately before the addition of hypochlorite and initiation of sonication (t = 0) then at 0.5, 1, 2, 5, 10, and 20 minutes. All experiments were performed at 20° C and the pH was monitored throughout. Acoustic input powers were calculated by calorimetry (Table 1). At the end of each reaction the effects of hypochlorite were stopped through addition of a 1% solution of sodium thiosulphate (Na 2 S 2 O 3 ). Table 1. Calorimetry Results Frequency Power Volume Diameter dT/Dt Power Intensity Energy Density Energy Dosage kHz Setting cm 3 cm 2 W W/cm 2 W/cm 3 Ws (J) 850 1 200 7.3 0.00155 1.2958 0.0310 0.0065 77.75 850 3 200 7.3 0.0231 19.3116 0.4616 0.0966 1158.70 20 40 200 1.3 0.0256 21.4016 16.1321 0.1070 1284.10 20 10 200 1.3 0.0038 3.1768 2.3946 0.0159 190.61 Conversely for simultaneous treatment the best kill (99.99%) is achieved using 20kHz at 21.4W whereas using 850kHz at 1.3W a much lower kill of 90% is obtained see Figure 2. When the power at 20kHz was reduced to a similar level to 850kHz (3.18W) the kill rate was reduced to 99% I.e. still significantly better. By increasing the power at 850kHz to a similar level to 20kHz (19.31W) the kill rate was only improved to 90%. A comparison of simultaneous treatment with ultrasound A comparison of pre-treatment with ultrasound Figure 1 Figure 2 Conclusion As previously shown there is a remarkable frequency effect in the use of ultrasound for disinfection using hyperchlorite. Pre-treatment is best at the higher frequency of 850kHz using a low power (1.3W) while simultaneous treatment is best at the lower frequency of 20kHz using a high power (21.4W). By increasing the power of the higher frequency the efficiency of kill was slightly retarded in pre-treatment and increased for simultaneous treatment. On the other hand decreasing the power of the lower frequency produced a slightly better kill when used in pre-treatment but a diminished effect in simultaneous treatment. The results also demonstrate that at similar powers the higher frequency is best applied as a pre-treatment and the lower frequency as a simultaneous treatment. These experiments show that under the conditions employed acoustic frequency was the main effect on the kill rate, depending upon whether pre-sonication or simultaneous treatment is employed acoustic power also has an influence, though less dramatic than frequency This suggests that in future work both factors will need to be optimised. Acknowledgement Helen Duckhouse thanks EPSRC for a Case Award in conjunction with SPT Ltd. References  Wastewater Microbiology, second edition, G. Bitton, Wiley-Liss, 2000.  The development and evaluation of ultrasound for the treatment of bacterial suspensions. A study of frequency, power and sonication time on cultured bacillus species, Joyce, E., Phull, S.S. Lorimer, J.P. and Mason, T.J., Ultrasonics Sonochemistry 10, pp 315-318, (2003) ISSN: 1350 4177.  The effect of sonication on microbial disinfection using hypochlorite, H. Duckhouse, T.J. Mason, S.S. Phull, and J.P. Lorimer, Ultrasonics Sonochemistry 11, pp 173-176 (2004). Pre-treatment using 850kHz at 1.3W shows the best kill (99.99%) while 20kHz at 21.4W gives the smallest (95%) see Figure 1. When the power delivered at 850kHz is raised to a similar level to 20kHz (19.31W) the kill rate was 99.9% I.e. still significantly better than at the lower frequency. This suggests de-clumping is the primary cause of the improved biocidal efficiency through presonication at 850kHz. When the power delivered at 20kHz is reduced to a similar level to 850kHz (3.18W) the kill rate was improved to 99.5%, nearly to the same kill rate of the higher frequency. Sonochemistry Centre, Coventry University