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Ultrasound enhancement of biocide efficiency
Ultrasonics Sonochemistry 11 (2004) 323–326 www.elsevier.com/locate/ultsonch
Ultrasound enhancement of biocide efficiency q T.R. Bott b
a,*
, Liu Tianqing
b
a Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Chemical Engineering Research Institute, Dalian University of Technology, Dalian 116012, China
Accepted 3 July 2003 Available online 3 September 2003
Abstract In order to take account of the likely increase in costs of biocides in the light of increasing legislation and concern for the environment, there is a need to maximise the efficiency of biocides for the control of biofouling. The use of ultrasound in conjunction with biocides offers such an opportunity. Tests have been carried out using ultrasound generated at 20 kHz in conjunction with the oxidising biocide ozone, in a laboratory pilot plant, to investigate the effects of mutuality. The preliminary results reported in this paper suggest that the combined effect of ultrasound and the biocide is better than either separately employed. Clearly substantially more work is required in order to maximise effectiveness for minimum cost. 2003 Elsevier B.V. All rights reserved.
1. Introduction Fouling of heat exchanger surfaces represents a serious operating problem. The presence of these deposits leads to reduced heat transfer efficiency, increased energy for pumping, additional maintenance and lost production due to the necessity for cleaning the equipment. The consequence is to increase operating costs and hence a reduction in the profitability of the particular process. Cooling water systems which often use water from a natural source, are prone to fouling through the accumulation of micro-organisms on equipment surfaces. Unless steps are taken to reduce or eliminate this biofouling, severe operating problems can be the result. The traditional method of controlling biofouling is to use a biocide to kill the micro-organisms, perhaps in conjunction with a biodispersant that reduces the opportunity for cells to accumulate on surfaces within the cooling water system. q This paper was originally presented at Applications of Power Ultrasound in Physical and Chemical Processing (Usound3) Paris December 2001. * Corresponding author. Tel.: +44-121-4145306; fax: +44-1214145324. E-mail address: [email protected] (T.R. Bott).
1350-4177/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1350-4177(03)00159-7
It is often the case that after use as a coolant, the water is returned to the environment for example into a river, lake or to the sea. The presence of the added toxic chemicals in the discharge represents a risk for the environment. In response to public concern about this practice, legislation has been or will be, introduced in an effort to restrict the discharge of toxic chemicals. In addition there is legislation, like the recent European Directive [1] which imposes a severe control on the development of new biocides. Although so called environmentally friendly biocides have been and are being developed, their cost is relatively high compared to traditional biocides such as chlorine. It is imperative therefore, that biocide application is efficient, so that the quantity used is minimised, in the interests of the environment and the reduction of operating costs. It is not usual, because of the associated cost, to employ continuous dosing of biocide, but to have a programme of intermittent dosing usually determined by trial and error. The effectiveness of a biocide depends on its mass transfer from bulk water flow to the bulk biiofilm/water interface. Mass transfer depends not only on the concentration gradient of the biocide between the bulk liquid and the biofouling, but also on the level of turbulence that facilitates mixing and transportation in the flowing water. For a given level of control the concentration of
324
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
the additive chemical can be reduced by increasing the turbulence in the system. Turbulence may be enhanced by different techniques, including the use of ultrasound. Increased turbulence itself can be effective in biofouling control. It is well known that increased shear produced by increased velocity can reduce the incidence of fouling [2]. Experimental work [3,4] has demonstrated the value of ultrasound alone for the control of biofouling in tubes representing the tubes in shell and tube heat exchangers. A logical extension, the subject of this paper, is to combine the use of ultrasound with low concentrations of biocide to provide enhanced effective biofouling control in flowing systems. The potential advantages were established by Ahmed and Russell [5] demonstrating that a combination of the effects of H2 O2 as a biocide and ultrasound had sporicidal and fungicidal properties not present in either H2 O2 treatment or ultrasound application alone. Furthermore it has been shown [6], that ultrasound enhanced the biocidal effects of the antibiotic gentamicin on Pseudomonas aeroginosa, on a microscope slide.
2. Experimental work A laboratory pilot plant designed to investigate biofouling using simulated cooling water, was used to assess the potential benefits of using ultrasound in conjunction with the application of a biocide. A single species of bacteria Pseudomonas fluorescens was used as the micro-organism in the simulated cooling water, grown in a fermenter of 5 l capacity. The operating conditions in terms of pH, nutrient availability, temperature and sterile air of the fermenter were carefully controlled to maintain a cell concentration of 1 · 109 cells. The residence time in the system was 30 min. The Pseudomonas species is a known slime former and is common in industrial cooling water systems, which make it a suitable organism for the tests. It is difficult to maintain the consistency of a multi-micro-organism system, which would be essential for a perfect representation of an industrial cooling water system. A simplified diagram of the laboratory pilot plant is shown in Fig. 1. The basis of the operation is a ‘‘feed and bleed’’ system. The simulated cooling water is, in effect, prepared in the mixing vessel. The water is filtered tap water, to remove the bacteria and other contaminating particles down to 0.2 lm. An activated carbon filter is used to remove all traces of chlorine from the water. A suspension of cells from the fermenter, is fed into the mixing vessel to provide a concentration of 1 · 106 cell/ml in the recirculating water. The pH is maintained at 7 by the automatic injection of 2 M K OH solution. Filtered air is bubbled into the mixing vessel since the Pseudomonas species is aerobic. Nutrients in fixed concentration in water, are pumped into the mix-
Fig. 1. Principle of pilot plant.
ing vessel at a known rate to maintain steady conditions. The residence time in these tests was 30 min. The experiments did not investigate any effects due to temperature. It is not anticipated that there would be any significant effect of temperature over the range of temperatures of the cooling water i.e. 25–30 C. The contaminated water from the mixing vessel is circulated through six vertical glass tubes 18 mm ID · 1 m in length representing the tubes in a shell and tube heat exchanger, at a velocity of 1 m/s. Although glass is not a common material of construction in a process plant it is satisfactory for the comparisons of biofilm accumulation required in this work, using a non-intrusive monitor developed for the purpose [7]. It is possible to correlate biofilm thickness from the infrared absorbance data by direct weighing, for given operating conditions. Assuming an even distribution of biofilm on the inner surface of the tubes, and that the biofilm has the density of water (biofilms are 90–95% water), it is possible to estimate the mean biofilm thickness from the total mass of biofilm accumulation. Two of the vertical tubes are fitted at their base, with a 600 W ultrasound processor (Vitracell, Sonics and Materials Inc.), to convert the 50 kHz power supply to high frequency electrical energy at 20 kHz which is transmitted to a piezolectic transducer to provide axially propagated ultrasound along the length of the tube. The density of the application based on the cross section of the tubes and the power rating of the processor was therefore 2357.8 kW/m2 . The biocide chosen for this experimental work was ozone. It is produced as required using a ‘‘Labo’’ generator (supplied by Ozotech Ltd.). Ozone laden air from the generator is bubbled through a vessel containing water, to provide a solution for use as a biocide. The exhaust air from the absorber is first passed through an ozone trap containing KI solution before discharge outside the laboratory. The concentration of ozone in the water in the absorber may be determined using iodimetric titration [8]. When required for the experiments the ozone solution is pumped into the mixing
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
vessel to maintain a fixed concentration in the circulating water. Before each experiment the whole apparatus is steam sterilised to ensure that there is no prior microbial contaminants. The nutrients are sterilised in an autoclave before use in the experiments.
325
Table 2 Mean Biofilm thickness over a period of 28 days with different treatment Week 2 3 4
Biofilm thickness for different tubes lm Ozone only
Ozone + ultrasound
26, 18, 17, 18 43, 15, 24, 20 111, 49, 60, 51
9, 8 11, 12 7, 5
3. Results and discussion Control experiments were carried out without any treatment, and also using ultrasound and ozone separately, before their combined use was investigated. 3.1. Ultrasound alone The initial study employed the 20 kHz ultrasound for one minute three times equally spaced throughout each 24-h period. Two test tubes were ‘‘dosed’’ at 20% amplitude for a period of 14 days. The results are summarised in Table 1. During the tests the mean biofilm thickness in the four tubes without ultrasound treatment i.e. no treatment whatsoever, was 46, 40, 31 and 56 lm respectively. The results indicate approaching 70% reduction in biofilm accumulation by using the ultrasound under the conditions of the experiments. Although there was some evidence that the inner surface of the tubes remote from the ultrasound source, were less efficiently cleaned, the mean biofilm thickness was lower than when no ultrasound was applied. 3.2. Combined use of ultrasound and ozone The basis of these experiments over a four-week period, was to allow biofilms to develop for one week under the flow conditions, with no treatment of any kind. The biofilm thickness developed in this period, for all six tubes was about 50 lm (45–60 lm). In the second week water containing ozone at concentrations of 2.2 mg/l was pumped into the system for 3 h each day, followed by ultrasound at 20% amplitude to two tubes for 3 min each day. In the third and fourth weeks the ozone concentration was raised from 2.2 to 2.8 mg/l. The data are presented in Table 2. No data on the bulk concentration of ozone was obtained, but the dilution
Table 1 Mean biofilm thickness over fourteen days operation Ultrasound amplitude
20%
Average biofilm thickness lm Before treatment
After first treatment
After second treatment
After third treatment
24
23
19
16
effect of the bulk circulating water, would make the concentration very low. It has been demonstrated [9] that concentration as low as 0.2 mg/l under similar conditions to the current experiments, removed substantial amounts of biofilm. Although there is scatter in the results the general conclusion is that the combined use of ozone and ultrasound under the conditions of the experiments, is more effective than either ozone or ultrasound alone. The data presented in Table 2 may be compared with the biofilm thickness of about 50 lm before any treatment was applied. Although there is a reduction of biofilm thickness due to the application of ozone, the removal is enhanced by the use of ultrasound. In general the removal of biofilm using either ozone or ultrasound alone was, in these particular experiments, similar.
4. Concluding remarks In cooling water systems the major effect of biofilm accumulation is to reduce the efficiency of the associated heat exchangers. Treatment of the cooling water therefore, is primarily aimed at removing the biofilm from the heat transfer surfaces. It does not necessarily mean that all the bacteria removed from the surfaces are killed, by the application of biocides although planktonic cells are very vulnerable to added biocide chemicals to the water. It could be argued that the application of ozone, and the subsequent oxidation of cell material provides nutrients for biofilm growth, but in industrial cooling water systems these nutrients would be removed from the system by the regular use of ‘‘blow down’’. They results do show that there is enhancement of ozone biocidal efficiency through the application of ultrasound. It is anticipated that similar results would be obtained for other biocides. Clearly considerably more research and development work is required to determine the optimum conditions i.e. ultrasound frequency, biocide dose level, and the periodically of both ultrasound and biocide application for given situations. Optimal conditions are likely to be different for different biocides and different operating conditions. There is also the need to compare the combined cost of biocide and ultrasound treatment in
326
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
relation to the use of any particular biocide and any necessary treatment of the water before final discharge. References [1] Anon,Directive98/8/ECoftheEuropeanParliamentandoftheCouncil, concerning the placing of biocidal products on the market. Official journalL123, 24/04/1998p0001-0063,16February1998. [2] L.F. Melo, T.R. Bott, Biofouling in water systems, Exp. Therm. Fluid Sci. 14 (4) (1997) 375–381. [3] I.E.C. Mott, D.J. Stickler, W.T. Coakley, T.R. Bott, The removal of bacterial biofilm from water filled tubes using axially propagated ultrasound, J. Appl. Microbiol. 84 (1998) 509–514. [4] T.R. Bott, Biofouling control with ultrasound, Heat Trans. Eng. 21 (3) (2000) 43–49.
[5] F.I.K. Ahmed, C. Russell, Synergism between ultrasonic waves and hydrogen peroxide in killing micro organisms, J. Appl. Bact. 39 (1975) 31–40. [6] Z. Qian, P. Stoodley, W.G. Pitt, Effect of low intensity ultrasound upon structure from confocal laser mircoscopy observation, Biomaterials 17 (1996) 1975–1980. [7] G. Bartlett et al., Measurement of biofilm development within flowing water using infrared absorbance, in: T.R. Bott et al. (Eds.), Understanding Heat Exchanger Fouling and its Mitigation, United Engng. Found. and Begelle House Inc., New York, 1999, pp. 337– 342. [8] Anon, Ozone for the treatment of potable water––laboratory procedures for assessing the dose rate, Ozotech Ltd 1984, 0T32. [9] K. Kaur et al., Effect of ozone on Pseudomonas fluorescens, in: L.F. Melo et al. (Eds.), Biofilms Science and Technology, Kluwer Academic Publishers, Dordrecht, 1992, pp. 589–594.
Ultrasound enhancement of biocide efficiency q T.R. Bott b
a,*
, Liu Tianqing
b
a Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Chemical Engineering Research Institute, Dalian University of Technology, Dalian 116012, China
Accepted 3 July 2003 Available online 3 September 2003
Abstract In order to take account of the likely increase in costs of biocides in the light of increasing legislation and concern for the environment, there is a need to maximise the efficiency of biocides for the control of biofouling. The use of ultrasound in conjunction with biocides offers such an opportunity. Tests have been carried out using ultrasound generated at 20 kHz in conjunction with the oxidising biocide ozone, in a laboratory pilot plant, to investigate the effects of mutuality. The preliminary results reported in this paper suggest that the combined effect of ultrasound and the biocide is better than either separately employed. Clearly substantially more work is required in order to maximise effectiveness for minimum cost. 2003 Elsevier B.V. All rights reserved.
1. Introduction Fouling of heat exchanger surfaces represents a serious operating problem. The presence of these deposits leads to reduced heat transfer efficiency, increased energy for pumping, additional maintenance and lost production due to the necessity for cleaning the equipment. The consequence is to increase operating costs and hence a reduction in the profitability of the particular process. Cooling water systems which often use water from a natural source, are prone to fouling through the accumulation of micro-organisms on equipment surfaces. Unless steps are taken to reduce or eliminate this biofouling, severe operating problems can be the result. The traditional method of controlling biofouling is to use a biocide to kill the micro-organisms, perhaps in conjunction with a biodispersant that reduces the opportunity for cells to accumulate on surfaces within the cooling water system. q This paper was originally presented at Applications of Power Ultrasound in Physical and Chemical Processing (Usound3) Paris December 2001. * Corresponding author. Tel.: +44-121-4145306; fax: +44-1214145324. E-mail address: [email protected] (T.R. Bott).
1350-4177/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1350-4177(03)00159-7
It is often the case that after use as a coolant, the water is returned to the environment for example into a river, lake or to the sea. The presence of the added toxic chemicals in the discharge represents a risk for the environment. In response to public concern about this practice, legislation has been or will be, introduced in an effort to restrict the discharge of toxic chemicals. In addition there is legislation, like the recent European Directive [1] which imposes a severe control on the development of new biocides. Although so called environmentally friendly biocides have been and are being developed, their cost is relatively high compared to traditional biocides such as chlorine. It is imperative therefore, that biocide application is efficient, so that the quantity used is minimised, in the interests of the environment and the reduction of operating costs. It is not usual, because of the associated cost, to employ continuous dosing of biocide, but to have a programme of intermittent dosing usually determined by trial and error. The effectiveness of a biocide depends on its mass transfer from bulk water flow to the bulk biiofilm/water interface. Mass transfer depends not only on the concentration gradient of the biocide between the bulk liquid and the biofouling, but also on the level of turbulence that facilitates mixing and transportation in the flowing water. For a given level of control the concentration of
324
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
the additive chemical can be reduced by increasing the turbulence in the system. Turbulence may be enhanced by different techniques, including the use of ultrasound. Increased turbulence itself can be effective in biofouling control. It is well known that increased shear produced by increased velocity can reduce the incidence of fouling [2]. Experimental work [3,4] has demonstrated the value of ultrasound alone for the control of biofouling in tubes representing the tubes in shell and tube heat exchangers. A logical extension, the subject of this paper, is to combine the use of ultrasound with low concentrations of biocide to provide enhanced effective biofouling control in flowing systems. The potential advantages were established by Ahmed and Russell [5] demonstrating that a combination of the effects of H2 O2 as a biocide and ultrasound had sporicidal and fungicidal properties not present in either H2 O2 treatment or ultrasound application alone. Furthermore it has been shown [6], that ultrasound enhanced the biocidal effects of the antibiotic gentamicin on Pseudomonas aeroginosa, on a microscope slide.
2. Experimental work A laboratory pilot plant designed to investigate biofouling using simulated cooling water, was used to assess the potential benefits of using ultrasound in conjunction with the application of a biocide. A single species of bacteria Pseudomonas fluorescens was used as the micro-organism in the simulated cooling water, grown in a fermenter of 5 l capacity. The operating conditions in terms of pH, nutrient availability, temperature and sterile air of the fermenter were carefully controlled to maintain a cell concentration of 1 · 109 cells. The residence time in the system was 30 min. The Pseudomonas species is a known slime former and is common in industrial cooling water systems, which make it a suitable organism for the tests. It is difficult to maintain the consistency of a multi-micro-organism system, which would be essential for a perfect representation of an industrial cooling water system. A simplified diagram of the laboratory pilot plant is shown in Fig. 1. The basis of the operation is a ‘‘feed and bleed’’ system. The simulated cooling water is, in effect, prepared in the mixing vessel. The water is filtered tap water, to remove the bacteria and other contaminating particles down to 0.2 lm. An activated carbon filter is used to remove all traces of chlorine from the water. A suspension of cells from the fermenter, is fed into the mixing vessel to provide a concentration of 1 · 106 cell/ml in the recirculating water. The pH is maintained at 7 by the automatic injection of 2 M K OH solution. Filtered air is bubbled into the mixing vessel since the Pseudomonas species is aerobic. Nutrients in fixed concentration in water, are pumped into the mix-
Fig. 1. Principle of pilot plant.
ing vessel at a known rate to maintain steady conditions. The residence time in these tests was 30 min. The experiments did not investigate any effects due to temperature. It is not anticipated that there would be any significant effect of temperature over the range of temperatures of the cooling water i.e. 25–30 C. The contaminated water from the mixing vessel is circulated through six vertical glass tubes 18 mm ID · 1 m in length representing the tubes in a shell and tube heat exchanger, at a velocity of 1 m/s. Although glass is not a common material of construction in a process plant it is satisfactory for the comparisons of biofilm accumulation required in this work, using a non-intrusive monitor developed for the purpose [7]. It is possible to correlate biofilm thickness from the infrared absorbance data by direct weighing, for given operating conditions. Assuming an even distribution of biofilm on the inner surface of the tubes, and that the biofilm has the density of water (biofilms are 90–95% water), it is possible to estimate the mean biofilm thickness from the total mass of biofilm accumulation. Two of the vertical tubes are fitted at their base, with a 600 W ultrasound processor (Vitracell, Sonics and Materials Inc.), to convert the 50 kHz power supply to high frequency electrical energy at 20 kHz which is transmitted to a piezolectic transducer to provide axially propagated ultrasound along the length of the tube. The density of the application based on the cross section of the tubes and the power rating of the processor was therefore 2357.8 kW/m2 . The biocide chosen for this experimental work was ozone. It is produced as required using a ‘‘Labo’’ generator (supplied by Ozotech Ltd.). Ozone laden air from the generator is bubbled through a vessel containing water, to provide a solution for use as a biocide. The exhaust air from the absorber is first passed through an ozone trap containing KI solution before discharge outside the laboratory. The concentration of ozone in the water in the absorber may be determined using iodimetric titration [8]. When required for the experiments the ozone solution is pumped into the mixing
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
vessel to maintain a fixed concentration in the circulating water. Before each experiment the whole apparatus is steam sterilised to ensure that there is no prior microbial contaminants. The nutrients are sterilised in an autoclave before use in the experiments.
325
Table 2 Mean Biofilm thickness over a period of 28 days with different treatment Week 2 3 4
Biofilm thickness for different tubes lm Ozone only
Ozone + ultrasound
26, 18, 17, 18 43, 15, 24, 20 111, 49, 60, 51
9, 8 11, 12 7, 5
3. Results and discussion Control experiments were carried out without any treatment, and also using ultrasound and ozone separately, before their combined use was investigated. 3.1. Ultrasound alone The initial study employed the 20 kHz ultrasound for one minute three times equally spaced throughout each 24-h period. Two test tubes were ‘‘dosed’’ at 20% amplitude for a period of 14 days. The results are summarised in Table 1. During the tests the mean biofilm thickness in the four tubes without ultrasound treatment i.e. no treatment whatsoever, was 46, 40, 31 and 56 lm respectively. The results indicate approaching 70% reduction in biofilm accumulation by using the ultrasound under the conditions of the experiments. Although there was some evidence that the inner surface of the tubes remote from the ultrasound source, were less efficiently cleaned, the mean biofilm thickness was lower than when no ultrasound was applied. 3.2. Combined use of ultrasound and ozone The basis of these experiments over a four-week period, was to allow biofilms to develop for one week under the flow conditions, with no treatment of any kind. The biofilm thickness developed in this period, for all six tubes was about 50 lm (45–60 lm). In the second week water containing ozone at concentrations of 2.2 mg/l was pumped into the system for 3 h each day, followed by ultrasound at 20% amplitude to two tubes for 3 min each day. In the third and fourth weeks the ozone concentration was raised from 2.2 to 2.8 mg/l. The data are presented in Table 2. No data on the bulk concentration of ozone was obtained, but the dilution
Table 1 Mean biofilm thickness over fourteen days operation Ultrasound amplitude
20%
Average biofilm thickness lm Before treatment
After first treatment
After second treatment
After third treatment
24
23
19
16
effect of the bulk circulating water, would make the concentration very low. It has been demonstrated [9] that concentration as low as 0.2 mg/l under similar conditions to the current experiments, removed substantial amounts of biofilm. Although there is scatter in the results the general conclusion is that the combined use of ozone and ultrasound under the conditions of the experiments, is more effective than either ozone or ultrasound alone. The data presented in Table 2 may be compared with the biofilm thickness of about 50 lm before any treatment was applied. Although there is a reduction of biofilm thickness due to the application of ozone, the removal is enhanced by the use of ultrasound. In general the removal of biofilm using either ozone or ultrasound alone was, in these particular experiments, similar.
4. Concluding remarks In cooling water systems the major effect of biofilm accumulation is to reduce the efficiency of the associated heat exchangers. Treatment of the cooling water therefore, is primarily aimed at removing the biofilm from the heat transfer surfaces. It does not necessarily mean that all the bacteria removed from the surfaces are killed, by the application of biocides although planktonic cells are very vulnerable to added biocide chemicals to the water. It could be argued that the application of ozone, and the subsequent oxidation of cell material provides nutrients for biofilm growth, but in industrial cooling water systems these nutrients would be removed from the system by the regular use of ‘‘blow down’’. They results do show that there is enhancement of ozone biocidal efficiency through the application of ultrasound. It is anticipated that similar results would be obtained for other biocides. Clearly considerably more research and development work is required to determine the optimum conditions i.e. ultrasound frequency, biocide dose level, and the periodically of both ultrasound and biocide application for given situations. Optimal conditions are likely to be different for different biocides and different operating conditions. There is also the need to compare the combined cost of biocide and ultrasound treatment in
326
T.R. Bott, L. Tianqing / Ultrasonics Sonochemistry 11 (2004) 323–326
relation to the use of any particular biocide and any necessary treatment of the water before final discharge. References [1] Anon,Directive98/8/ECoftheEuropeanParliamentandoftheCouncil, concerning the placing of biocidal products on the market. Official journalL123, 24/04/1998p0001-0063,16February1998. [2] L.F. Melo, T.R. Bott, Biofouling in water systems, Exp. Therm. Fluid Sci. 14 (4) (1997) 375–381. [3] I.E.C. Mott, D.J. Stickler, W.T. Coakley, T.R. Bott, The removal of bacterial biofilm from water filled tubes using axially propagated ultrasound, J. Appl. Microbiol. 84 (1998) 509–514. [4] T.R. Bott, Biofouling control with ultrasound, Heat Trans. Eng. 21 (3) (2000) 43–49.
[5] F.I.K. Ahmed, C. Russell, Synergism between ultrasonic waves and hydrogen peroxide in killing micro organisms, J. Appl. Bact. 39 (1975) 31–40. [6] Z. Qian, P. Stoodley, W.G. Pitt, Effect of low intensity ultrasound upon structure from confocal laser mircoscopy observation, Biomaterials 17 (1996) 1975–1980. [7] G. Bartlett et al., Measurement of biofilm development within flowing water using infrared absorbance, in: T.R. Bott et al. (Eds.), Understanding Heat Exchanger Fouling and its Mitigation, United Engng. Found. and Begelle House Inc., New York, 1999, pp. 337– 342. [8] Anon, Ozone for the treatment of potable water––laboratory procedures for assessing the dose rate, Ozotech Ltd 1984, 0T32. [9] K. Kaur et al., Effect of ozone on Pseudomonas fluorescens, in: L.F. Melo et al. (Eds.), Biofilms Science and Technology, Kluwer Academic Publishers, Dordrecht, 1992, pp. 589–594.