Ultrasound enhancement of near-neutral photo-Fenton for effective E. coli inactivation in wastewater

1  Ultrasound enhancement of near-neutral photo-Fenton for effective E. coli 1 inactivation in wastewater 2 3 Stefanos Giannakis1,2,3, Stefanos Papoutsakis3, Efthymios Darakas1, Antoni Escalas-4 Cañellas2,4, Christian Pétrier5,6, César Pulgarin3,* 5 1Laboratory of Environmental Engineering and Planning, Department of Civil Engineering, Aristotle University of 6 Thessaloniki, 54624 Thessaloniki, Greece 7 2Laboratory of Control of Environmental Contamination, Institute of Textile Research and Industrial Cooperation of Terrassa 8 (INTEXTER), Universitat Politècnica de Catalunya, Colom 15, 08222 Terrassa, Catalonia, Spain 9 3Swiss Federal Institute of Technology, Lausanne, Institute of Chemical Sciences and Engineering, 1015 Lausanne, 10 Switzerland 11 4Department of Chemical Engineering & Terrassa School of Engineering, Universitat Politècnica de Catalunya, Colom 1, 12 08222, Terrassa, Catalonia, Spain 13 5Laboratoire Rhéologie et Procédés, Grenoble-INP, UMR CNRS 5520, BP 53, 38041 Grenoble Cedex 9, France 14 6King AbdulAziz University, Jeddah, Saudi Arabia 15 *Corresponding author: César Pulgarin, Tel: +41216934720; Fax: +41216936161; E-mail: 16 [email protected] 17 18 Abstract 19 In this study, we attempt for the first time to couple sonication and photo-Fenton for bacterial 20 inactivation of secondary treated effluent. Synthetic wastewater was subjected to sequential high-21 frequency/low power sonication, followed by mild photo-Fenton treatment, under a solar simulator. It 22 was followed by the assessment of the contribution of each component of the process (Fenton, US, hv) 23 towards the removal rate and the long-term survival; sunlight greatly improved the treatment 24 efficiency, with the coupled process being the only one to yield total inactivation within the 4-h period 25 of treatment. The short-term beneficial disinfecting action of US and its detrimental effect on bacterial 26 survival in long term, as well as the impact of light addition were also revealed. Finally, an 27 investigation on the operational parameters of the process was performed, to investigate possible 28 improvement and/or limitations of the coupled treatment; 3 levels of each parameter involved 29 (hydraulic, environmental, US and Fenton) were tested. Only H2O2 increased improved the process 30 significantly, but the action mode of the joint process indicated potential cost-effective solutions 31 towards the implementation of this method. 32 Keywords: wastewater disinfection, photo-Fenton, high-frequency ultrasound, E. coli, inactivation 33 mechanism34 NOTICE:thisistheauthor’sversionofaworkthatwasacceptedforpublicationinUltrasonicsSonochemistr y .Changesresultingfromthepublishingprocess,suchas peerreview,editing,corrections,structuralformatting,andotherqualitycontrolmechanismsmaynotbereflectedinthisdocument.Changesmayhavebeenmadeto thisworksinceitwassubmittedforpublication.AdefinitiveversionwassubsequentlypublishedinUltrasonicsSonochemistry[Vol.22,p.515‐526,January2015].DOI: 10.1016/j.ultsonch.2014.04.015 2  1. INTRODUCTION 35 36 Advanced Oxidation Processes (AOPs) have been in the spotlight for more than three decades, as part 37 of a global effort to modernize actual methods of water disinfection. Their action is based on the 38 production of the extremely oxidizing hydroxyl radical (•OH) [1], which can attack the chemical 39 structure of the microorganisms’ cell wall and inactivate them [2]. Ultrasound has been extensively 40 studied as an AOP, targeting microorganism inactivation, such as bacteria, viruses etc., by either low 41 (~20 kHz) or high frequencies (200+ kHz) [2, 3, 4 and 5]. This method is exploiting the direct 42 mechanical action of the cavitation bubble implosion (low frequencies) as well as the additional 43 production of H2O2 and •OH radicals during cavitation (high frequencies); the propagation of 44 ultrasound waves in the aqueous medium initiates the aforementioned actions, by the generation of 45 extreme temperature and pressure conditions [6], which have a proven bactericidal effect [7, 8, 9, and 46 10]. 47 As far as the ultrasound set-up is concerned, the frequency of the ultrasonic waves is a crucial 48 parameter, for it defines the size of the cavitation bubbles [11]. Literature suggests that the average 49 cavity size is proportional to the acoustic power and inversely proportional to the ultrasound frequency 50 [12]. It is also verified that apart from low frequency/high power ultrasound systems [13, 14], high 51 frequency/low power processes have been proven to efficiently inactivate microorganisms [8, 15, and 52 16]. However, ultrasound already requires high intensities to achieve total inactivation of 53 microorganisms, and therefore, is considered an expensive application for large volumes of water [2]. 54 Considering all the above, it should be used preferably as a complementary disinfecting method [2]. 55 The photo-Fenton process [17] could play the role of the main disinfecting method, as one of the most 56 efficient methods of hydroxyl radical production [18]. Lately, it has even been used to disinfect 57 drinking water, being a good alternative to chlorination, with its known disinfection by-products 58 formation [19]. However, wastewater is a complex matrix in which many organic and inorganic 59 compounds coexist, such as nutrients, salts and many substances that could influence the outcome of 60 the application of either process. It has been reported that the presence of hydroxyl radical scavengers, 61 namely the organic matter, presents an additional oxidation target and renders AOPs sensitive to the 62 treatment of wastewater [20, 21]. Suppression of these scavengers revealed their importance [10] and 63 also, for years the Fenton reaction was believed to be a pH-restricted reaction in highly acidic regions; 64 it was considered impossible to apply such methods, in matrices with near-neutral pH [22]. However, 65 recent advances [18, 23 and 24] have proven its effectiveness in the neutral area, and in the 66 simultaneous presence of organic matter [18, 25]. Previous work in our group has shown, there is no 67 need for acidification prior to the treatment to keep a significant part of the iron soluble; apart from the 68 direct complexion with bacteria, there are some strong photoactive Fe3+ complexes formed in presence 69 of organic matter [21, 25]: 70 3  [R-COO- - Fe3+]2+  hv  Fe2+ + CO2 + R• (1) 71 The cycle continues with the reaction of the regenerated iron with hydrogen peroxide to produce more 72 hydroxyl radicals etc. 73 Fe2+ + H2O2  OH- + •OH + Fe3+ (2) 74 In order to increase the amount of water treated by solar-assisted methods, compound parabolic 75 collector reactors have been used [17, 18, 23 and 26], and solar photo-Fenton even was a subject under 76 question, because of the intermittent action of the light [27]. There is a technical issue to be addressed 77 in the intermittent nature of this treatment method, and the existence of “dead” time among the 78 experiment. Typically, a CPC photo-reactor consists of the illuminated surface and the storage-79 recirculation tank. The recirculating flow of these reactors creates a gap in the illumination for as long 80 as water is present in the (dark) storage tank, allowing bacterial defense mechanisms to deploy [28]. 81 Literature indicates a variety of light-to-dark distributions (Table 1), which materialize this difference 82 [17, 18, 23, 26, 29, 30 and 31]. 83 Therefore, keeping in mind the improvement of the near-neutral photo-Fenton disinfection while 84 working within realistic operational parameters, for the first time we study the joint ultrasound/photo-85 Fenton treatment for wastewater, in a CPC-like, lab-scale system. In this manner, we will take 86 advantage of two factors that could work complementing each other: firstly, the exploitation of the 87 dark intervals for sonication, along with the utilization of solar energy for the promotion of a mild 88 photo-Fenton reaction and secondly, the supplementary action these processes have, since, for 89 instance, US can produce H2O2 and subsequently, could fuel the photo-Fenton process. In our study, 90 synthetic secondary effluent was used, spiked with E. coli K12, recirculating around a sonicated dark 91 reactor and an illuminated batch reactor, under solar simulated light. We aim to: 92 i) Explore the effects of the photo-Fenton factors (light, reactants) and the ultrasonic action 93 (US) on both short and long-term disinfection events; clarification of the effects is 94 attempted by stepwise insertion of the participating actions. 95 ii) Investigate the involved operational parameters (recirculation speed, temperature, light 96 intensity, treated volume and distribution of volumes, iron and hydrogen peroxide content, 97 ultrasound intensity) in a small-scale set-up. 98 99 2. MATERIALS AND METHODS 100 101 2.1. Synthetic secondary effluent preparation 102 103 4  2.1.1. Microbial methods 104 The E. coli strain K12 (MG1655) employed was provided by the “Deutsche Sammlung von 105 Mikroorganismen und Zellkulturen”. Luria-Bertani broth was inoculated with a colony from bacterial 106 E. coli pre-cultures, placed in 50 ml plastic falcons for 8 h and then loop inoculated, after 1% dilution 107 overnight (180 rpm and 37°C for 15 h), to achieve stationary phase cells. 108 Harvested cells were centrifuged and washed three times (5000 rpm, 15 and 5 min for separation and 109 washing, respectively), followed by reservation in saline solution (neutral pH solution with 8 g/L NaCl 110 and 0.8 g/L KCl); a solution of 109 CFU/mL is achieved. 111 112 2.1.2. Synthetic wastewater composition 113 The preparation of the synthetic wastewater took place as under the directive of SYMAWE [32]. The 114 initial DOC was 100 mg/L (250 mg/L COD). The experiments used a 10% dilution (in distilled water) 115 of the said composition. The dilution performed corresponds to the COD and DOC values encountered 116 in normal secondary effluents. Finally, the pH of the sample was between 6.5-7. 1 mL of the prepared 117 bacterial solution was used to spike the diluted wastewater, thus resulting in an initial bacterial 118 population of 106 CFU/mL. 119 120 2.2. Reagents and analyses 121 122 The wastewater constituents, as well as the Fenton reagents were used as received. Photo-Fenton 123 experiments were carried out employing ferrous sulfate heptahydrate (Fluka Chemika), hydrogen 124 peroxide (35% by weight, Sigma Aldrich), used as received. The dissolved iron (Fe2+, Fe3+) was 125 measured with the ferrozine method [33], using a UV-Vis Lambda 20 spectrophotometer, provided by 126 PerkinElmer, Schwerzenbach, Switzerland. For 1.6 mL of sample 0.2 of ferrozine solution (4.9 mM) 127 was added, followed by 0.2 mL of hydroxylamine hydrochloride solution 10% w/w. Acetate buffer 128 solution was added for a final 4.5-5 pH value. To determine the concentration of hydrogen peroxide in 129 the sample titanium oxysulfate solution was added, also measured with the same spectrophotometer. 130 The pH of each sample was measured with a pH-meter provided by Mettler Toledo (PH/Ion S220, 131 Seven Compact, Mettler Toledo). 132 133 2.3. Description of reactors’ set-up 134 135 The preliminary study was carried out in plain Pyrex glass batch reactors of 65 mL total capacity. In 136 the set-up presented in Figure 1, the configuration permits the sequential treatment of the synthetic 137 5  wastewater; US/photo-Fenton treatment was taking place (or vice versa). The same configuration was 138 used in one of our previous works, (used in [29], similar to the set-up used by Mendez-Arriaga et al, 139 2009 [34]), synthetic wastewater from a cylindrical double-wall glass vessel (400 mL) was pumped by 140 a peristaltic pump through three glass reactors (diameter 3.8 cm, effective irradiation surface 214.8 141 cm2), connected in series, of total volume 230 mL. Temperature was regulated by water recirculating 142 around the reactor and connected to a thermostat. The third reactor effluent was recirculated to the 143 original vessel. Normally, water was inserted in the double-wall reactor and pumped into the irradiated 144 part. Therefore, 230 mL of water were always present under illumination, 70 mL in the distribution 145 system and the rest subjected to sonication. 146 The ultrasonic waves (275 kHz) were emitted from a piezoelectric 4-cm disc, fixed on a Pyrex glass 147 plate adjusted to the bottom of the double-walled reactor. The intensities applied in all experiments 148 were 10, 20 and 40 W. The electric power was the chosen method to calibrate the ultrasonic 149 equipment. The in-series reactors were irradiated by the Suntest apparatus. The Suntest CPS solar light 150 simulator bears a lamp that emits ~0.5% of the photons at wavelengths <300 nm, ~7% between 300 151 and 400 nm and the rest follow the solar spectrum. The global irradiance values used in this work were 152 800, 1000 and 1200 W/m2, while the corresponding UV values were approximately 19.2, 24.7 153 and 30.2 W/m2. 154 155 2.4. Experimental design 156 157 Two sets of experiments were performed. In a first set of 8 experiments, that we call step-wise 158 construction of the joint treatment process, the elements of the US/hv/Fe/H2O2 were gradually and 159 accumulatively applied to the wastewater, in order to determine the individual role of each factor and 160 to detect any synergy among them. Table 2 shows the conditions corresponding to each individual 161 treatment factor when applied. Table 3 summarizes the four subsets of experiments in the step-wise 162 design. 163 In a second set of experiments (improvement of the process efficiency), eight different variables were 164 individually modified at three levels, while keeping the other variables constant, in order to obtain 165 improved working levels for each variable. Table 4 displays the three values (levels) essayed for each 166 variable. In each experiment the remaining parameters were kept constant and set to the central value 167 shown in the table. 168 169 2.5. Bacterial enumeration and regrowth tests 170 171 6  The disinfection efficiency was measured by viable plate counts on Petri dishes containing PCA agar 172 (plastic, 9-cm diameter). The pour-plating method was used and dilutions were made to ensure 173 countable numbers on the plates, i.e. 20-100 colonies/plate. Experiments were performed twice and 174 plating took place in 2-3 consequent dilutions and in duplicates. 175 Regrowth of bacteria was estimated after the storage of the samples at ambient temperature for 24 and 176 48 h after the sampling time. Samples were kept in 1.5 mL plastic Eppendorf caps in the dark and the 177 population was measured to assess the post-irradiation events, after their removal from the 178 experimental set-up. 179 180 3. RESULTS AND DISCUSSION 181 182 3.1. Results of the step-wise construction of the joint treatment process 183 184 As far as a potential application of mild photo-Fenton assisted by high frequency/low power 185 ultrasound is concerned, moderate concentrations of reactants are suggested for the evolution of our 186 study, after an initial investigation (data not shown). At 1000 W/m2 light intensity, an addition of 1 187 ppm iron and 10 ppm of H2O2 will be used, as marginal values of Fenton reagents and 20 W of US 188 power. 189 190 3.1.1. Disinfection efficiency 191 192 i) Experiments: 1-2 (WW and WW/Fe/H2O2). 193 Figure 2a presents the results of the first part of the experiments, where neither light nor US was 194 applied. Wastewater was recirculated around the non-illuminated, non-sonicated experimental set-up 195 and the corresponding graphs describe the changes when H2O2 and iron were added to the solution. 196 We notice the increase of the population, when no reactants were added, due to the existence of 197 nutrients and salts that favor bacterial growth in this water matrix [35]. H2O2 is a substance with 198 disinfecting action, while iron itself is not toxic for bacteria. The addition of both reactants causes the 199 initiation of the Fenton reaction, which has a slow, but existing disinfecting ability and within a 200 timeframe of 4 h, we observe a 24.4% reduction in the initial population. 201 202 ii) Experiments: 3-4 (US and US/Fe/H2O2). 203 7  Figure 2b demonstrates the effects sonication has on samples, alongside with the stepwise insertion of 204 the Fenton reagents. The sample recirculates around the ultrasound vessel and the non-illuminated 205 area, being subject to intermittent high-frequency, low intensity sonication. When ultrasound alone is 206 applied, there is a decrease in total bacterial numbers, approaching 27.9%. The concurrent addition of 207 both Fenton reactants (H2O2 and Fe2+) in the sonicated sample causes an 82.1% reduction in the 208 bacterial population, compared to 27.9 % reduction for US treatment and 24.4% for Fenton treatment 209 alone. This indicates a synergy between sonication and the Fenton reagents; a synergy factor of 1.57 is 210 demonstrated by the disinfecting efficiency of the reactions. 211 During sonication, the breakage of the cavitation bubbles can lead to the formation of an almost point-212 sized heat source [36, 37], with local temperatures approaching 2000 K and pressures of 200 atm. 213 These extreme conditions can cause lysis of water molecules and along with that, extra production of 214 hydroxyl radicals [8]. The presence of the afore-mentioned particles in real wastewater and the 215 bacteria (in our matrix) could also play another important role, since the collapse of the cavitation 216 bubbles near a particle in the medium could cause micro-jets, depending on the size of the particle [38] 217 and could also form “weak spots” in the body of the liquid; these are potential places to form a cavity 218 [39]. It has been also reported that the presence of some salts causes a baro-protective effect on the 219 cells [40] and samples with higher contents of soluble solids would require higher sonication times. 220 Apart from the physical damage, during the ultrasound treatment of the sample, there is ample 221 generation of reactive oxygen species (•OH radicals [41], singlet oxygen [42, 43]), as mentioned 222 before, which are known to stress bacteria and lead to cell death [25, 44]. 223 Finally, the addition of peptone (present in the synthetic wastewater) and the generally, presence of 224 nitrogen compounds has been reported to delay the sonicated degradation of phenols [45]. However, 225 nitrogen, under the presence of ultrasound waves can form NOx (nitrate and nitrite). Its reaction with 226 singlet oxygen (as produced before) [43] produces peroxynitrite (ONOO-) [46]. Peroxynitrite is 227 included in the reactive nitrogen species and can cause significant injures to various structures of the 228 cell (free radical damage or attack against the respiratory chain) [46]. 229 The synergistic action of US and Fenton processes can be attributed to the exploitation of the 230 recombined H2O2 (from •OH), which is less oxidative than the hydroxyl radical itself, and with that, the 231 re-initiation of the Fenton reaction with new reactants. Also, the ultrasound process, according to 232 Kryszczuk [47], increases the transient breakage of the bonds among the molecular components of the 233 cell membrane, which increases the permeability of the cell in external substances [48]. Therefore, the 234 introduction of Fe2+ in the cell is easier and its presence inside the cell can produce hydroxyl radicals 235 very close to vital functions of the cell, as well as the DNA [25] due to the induced internal Fenton 236 process. 237 238 iii) Experiments: 5-6 (hv and hv/Fe/H2O2). 239 8  The 3rd set of experiments is dedicated in the investigation of the impact of light in the sequential 240 process. In all experiments light is provided at 1000 W/m2, but in total, intermittent irradiation is 241 provided to the system; there is an illuminated regime and a non-illuminated one, in the Suntest 242 apparatus and the (inactive) sonication vessel (and tubing). In one of our previous works [29], we 243 demonstrated the impact light intermittence has on bacterial disinfection and survival, while 244 continuous supply or very fast recirculation around illuminated and dark regimes favors disinfection, 245 with the same set-up. Therefore, photo-Fenton is promoted in non-intermittent regimes or, as in our 246 case, short dark interval periods. 247 As it can be seen from Figure 2c, light, even in non-continuous form, is very effective and results in 248 high inactivation rates. Its disinfecting action is dominating the removal process, until the Fenton 249 reagents are present, and solar-assisted photo-Fenton is induced. The action of photo-Fenton is taking 250 place within the Suntest and dark (normal) Fenton takes place during the rest of the time, in a 0.85:1 251 time distribution (46% photo-Fenton over 54% Fenton). After an initial delay, which is demonstrated 252 as a shoulder in the graph, reaction is more effective by the hv/Fe/H2O2 than the corresponding solar 253 treatment. 254 Spuhler et al. [25] have reviewed the mechanism of bacterial inactivation by the photo-Fenton reaction 255 in near-neutral water with organic components, and have suggested the possible sources of ROS 256 production and cellular photo-oxidative damage, as well as the damage done by the ROS themselves, 257 deriving from the photo-Fenton reaction. In our suggested treatment method, these mechanisms are 258 completely compatible, explaining the majority of the actions and other works on near-neutral photo-259 Fenton mechanisms describe fully the mechanisms, so we will not further analyze their findings. 260 261 iv) Experiments: 7-8 (hv/US and hv/US/Fe/H2O2). 262 The final group of experiments are presented in Figure 2d. This graph summarizes the results of the 263 joint treatment by light and ultrasound. It is clear, after a comparison with Figure 2c, that when light is 264 present, its disinfecting action is dominating the process. However, we observe that the only case total 265 disinfection is achieved, is by the sequential US/pF system. In this system, wastewater spends its time 266 distributed 46% under photo-Fenton, 14% in the dark (dark-Fenton) and 40% in the sonication vessel 267 (US/Fenton). The experimental time has less inactive periods, and we observe that it has a significant 268 impact in the total inactivation of the bacterial populations in less than 4h. Here, the photo-Fenton/US 269 synergy is low in terms of bacterial counts, efficiency was improved in a relatively low percentage, but 270 only the coupled process resulted in total disinfection in 4 h. The elevated efficiency and total 271 inactivation for the first time, is attributed to the combination of all the previous actions (in US and/or 272 light), as well as the following actions (a graphical summary of all the actions is presented in Figure 273 3): 274 9  i. The hydroxyl radical is a short living ROS, and it occurs not to reach the target in all cases and 275 often recombines to create H2O2 [49]. Therefore, the addition of ultrasound directly produces 276 hydroxyl radicals and H2O2; the •OH directly attacks the cell and H2O2 participates in the 277 photo-Fenton reaction (2). Alongside with the added H2O2, there is additional production, 278 fueling the Fenton reaction and thus, improving the overall efficiency of the treatment. 279 ii. As we described before, with ultrasound waves, the loosening of transient bonds and insertion 280 of Fe2+ in the cell is increased, which promotes the internal Fenton reaction. After the 281 completion of the Fenton reaction, light reduces Fe3+ to Fe2+, and re-initiates a radical 282 production inside the cell (internal photo-Fenton). 283 iii. Low frequency ultrasound has been proven [50] to reduce Fe3+ in the form of ferrous ions 284 (Fe2+). The average size of the bubble however decreases when frequency is increased, in our 285 system [12]; nevertheless, cavitation still takes place. Therefore, it is possible that an action 286 like this could provide an additional source of iron available for the photo-Fenton process, and 287 progress the regeneration of the catalyst in the (otherwise) non-illuminated part of the time. In 288 that way, more available ferrous ions can be present in the solution. 289 iv. We mentioned the extreme temperature and pressure conditions that take place during the 290 collapse of the cavitation bubbles. The interior part of the bubble, under these conditions, is 291 known to emit light, under the phenomenon of sono-luminescence [8]. The optical aspects of 292 this phenomenon have been studied [51] and the emitted light wavelengths fall into the 293 necessary ones possibly able i) to induce the regeneration of the photo-Fenton reaction 294 catalyst, ii) inflict direct UV damage to the cell. However, the necessary energy to achieve this 295 is still under question. 296 v. Apart from the radicals’ production through the normal photo-Fenton cycle, the presence of 297 light is participating in another series of reactions with nitrogen compounds. The photolysis of 298 nitrate and nitrites (produced by the participation of the US in the process) can lead to 299 additional hydroxyl radical production [45]: 300 Photolysis of Nitrate: 301  →    (3) 302  →      (4) 303    ↔    (5) 304 Photolysis of Nitrite: 305  →     (6) 306    ↔    (7) 307 308 16  Swiss Government for the Swiss Government Excellence Scholarship, by means of which Stefanos 518 Giannakis has received a Research Visit fellowship (No. 2012.0499). Finally, Stefanos Papoutsakis 519 was funded by the Swiss-Hungarian Co-operation Program “Sustainable fine chemical, 520 pharmaceutical industry: screening and utilization of liquid wastes – Innovative approaches for the 521 abatement of industrial/toxic waste in aqueous effluents”. 522 523 6. REFERENCES 524 525 1. I. Oller, S. Malato, J.A. Sánchez-Pérez, W. Gernjak, M.I. Maldonado, L.A. Pérez-Estrada, C. 526 Pulgarín, A combined solar photocatalytic-biological field system for the mineralization of an 527 industrial pollutant at pilot scale, Catalysis Today, 122 (2007) 150-159. 528 2. A. Antoniadis, I. Poulios, E. Nikolakaki, D. Mantzavinos, Sonochemical disinfection of 529 municipal wastewater, Journal of hazardous materials, 146 (2007) 492-495. 530 3. P. Foladori, B. Laura, A. Gianni, Z. Giuliano, Effects of sonication on bacteria viability in 531 wastewater treatment plants evaluated by flow cytometry--fecal indicators, wastewater and activated 532 sludge, Water research, 41 (2007) 235-243. 533 4. C.V. Chrysikopoulos, I.D. Manariotis, V.I. Syngouna, Virus inactivation by high frequency 534 ultrasound in combination with visible light, Colloids and surfaces. B, Biointerfaces, 107 (2013) 174-535 179. 536 5. T.J. Mason, E. Joyce, S.S. Phull, J.P. Lorimer, Potential uses of ultrasound in the biological 537 decontamination of water, Ultrasonics sonochemistry, 10 (2003) 319-323. 538 6. Y.T. Didenko, W.B. McNamara, K.S. Suslick, Hot spot conditions during cavitation in water, 539 Journal of the American Chemical Society, 121 (1999) 5817-5818.. 540 7. I. Hua, M.R. Hoffmann, Optimization of ultrasonic irradiation as an advanced oxidation 541 technology, Environmental Science & Technology, 31 (1997) 2237-2243. 542 8. I. Hua, J.E. Thompson, Inactivation of Escherichia coli by sonication at discrete ultrasonic 543 frequencies, Water research, 34 (2000) 3888-3893. 544 9. M. Furuta, M. Yamaguchi, T. Tsukamoto, B. Yim, C. Stavarache, K. Hasiba, Y. Maeda, 545 Inactivation of Escherichia coli by ultrasonic irradiation, Ultrasonics sonochemistry, 11 (2004) 57-60. 546 10. M.F. Dadjour, C. Ogino, S. Matsumura, N. Shimizu, Kinetics of disinfection of Escherichia 547 coli by catalytic ultrasonic irradiation with TiO2, Biochemical Engineering Journal, 25 (2005) 243-548 248. 549 11. K.S. Suslick, Sonochemistry, Science, 247 (1990) 1439-1445. 550 12. A. Brotchie, F. Grieser, M. Ashokkumar, Effect of power and frequency on bubble-size 551 distributions in acoustic cavitation, Physical review letters, 102 (2009) 084302. 552 13. S. Drakopoulou, S. Terzakis, M. Fountoulakis, D. Mantzavinos, T. Manios, Ultrasound-553 induced inactivation of gram-negative and gram-positive bacteria in secondary treated municipal 554 wastewater, Ultrasonics sonochemistry, 16 (2009) 629-634. 555 14. D. Venieri, E. Markogiannaki, E. Chatzisymeon, E. Diamadopoulos, D. Mantzavinos, 556 Inactivation of Bacillus anthracis in water by photocatalytic, photolytic and sonochemical treatment, 557 Photochemical & Photobiological Sciences, (2013). 558 15. R.V. Peterson, W.G. Pitt, The effect of frequency and power density on the ultrasonically-559 enhanced killing of biofilm-sequestered Escherichia coli, Colloids and Surfaces B: Biointerfaces, 17 560 (2000) 219-227. 561 17  16. A. Moody, G. Marx, B.G. Swanson, D. Bermúdez-Aguirre, A comprehensive study on the 562 inactivation of Escherichia coli under nonthermal technologies: High hydrostatic pressure, pulsed 563 electric fields and ultrasound, Food Control, 37 (2014) 305-314. 564 17. A.-G. Rincón, C. Pulgarin, Fe3+and TiO2 solar-light-assisted inactivation of E. coli at field 565 scale: Implications in solar disinfection at low temperature of large quantities of water, Catalysis 566 today, 122 (2007) 128-136. 567 18. A. Moncayo-Lasso, J. Sanabria, C. Pulgarin, N. Benitez, Simultaneous E. coli inactivation and 568 NOM degradation in river water via photo-Fenton process at natural pH in solar CPC reactor. A new 569 way for enhancing solar disinfection of natural water, Chemosphere, 77 (2009) 296-300. 570 19. R.J. Bull, F.C. Kopfler, Health effects of disinfectants and disinfection by-products, AWWA, 571 1991. 572 20. T.M. Olson, P.F. Barbier, Oxidation kinetics of natural organic matter by sonolysis and ozone, 573 Water research, 28 (1994) 1383-1391. 574 21. E. Ortega-Gómez, M.M.B. Martín, B.E. García, J.A.S. Pérez, P.F. Ibáñez, Solar Photo-Fenton 575 for Water Disinfection: An Investigation of the Competitive Role of Model Organic Matter for 576 Oxidative Species, Applied Catalysis B: Environmental, (2013). 577 22. R.A. Torres, F. Abdelmalek, E. Combet, C. Petrier, C. Pulgarin, A comparative study of 578 ultrasonic cavitation and Fenton's reagent for bisphenol A degradation in deionised and natural waters, 579 Journal of hazardous materials, 146 (2007) 546-551. 580 23. F. Sciacca, J.A. Rengifo-Herrera, J. Wéthé, C. Pulgarin, Solar disinfection of wild Salmonella 581 sp. in natural water with a 18L CPC photoreactor: Detrimental effect of non-sterile storage of treated 582 water, Solar Energy, 85 (2011) 1399-1408. 583 24. J. Rodríguez-Chueca, M.I. Polo-López, R. Mosteo, M.P. Ormad, P. Fernández-Ibáñez, 584 Disinfection of real and simulated urban wastewater effluents using mild solar photo-Fenton, Applied 585 Catalysis B: Environmental. 586 25. D. Spuhler, J. Andrés Rengifo-Herrera, C. Pulgarin, The effect of Fe2+, Fe3+, H2O2 and the 587 photo-Fenton reagent at near neutral pH on the solar disinfection (SODIS) at low temperatures of 588 water containing Escherichia coli K12, Applied Catalysis B: Environmental, 96 (2010) 126-141. 589 26. P. Fernández, J. Blanco, C. Sichel, S. Malato, Water disinfection by solar photocatalysis using 590 compound parabolic collectors, Catalysis Today, 101 (2005) 345-352. 591 27. A.-G. Rincón, C. Pulgarin, Absence of E. coli regrowth after Fe3+and TiO2 solar 592 photoassisted disinfection of water in CPC solar photoreactor, Catalysis today, 124 (2007) 204-214. 593 28. D.B. Misstear, J.P. Murtagh, L.W. Gill, The Effect of Dark Periods on the UV Photolytic and 594 Photocatalytic Disinfection of Escherichia coli in a Continuous Flow Reactor, Journal of Solar Energy 595 Engineering, 135 (2013) 021012-021012. 596 29. S. Giannakis, A.I. Merino Gamo, E. Darakas, A. Escalas-Cañellas, C. Pulgarin, Impact of 597 different light intermittence regimes on bacteria during simulated solar treatment of secondary 598 effluent: Implications of the inserted dark periods, Solar Energy, 98, Part C (2013) 572-581. 599 30. J. Ndounla, S. Kenfack, J. Wéthé, C. Pulgarin, Relevant impact of irradiance (vs. dose) and 600 evolution of pH and mineral nitrogen compounds during natural water disinfection by photo-Fenton in 601 a solar CPC reactor, Applied Catalysis B: Environmental, 148–149 (2014) 144-153. 602 31. P. Fernández-Ibáñez, C. Sichel, M. Polo-López, M. de Cara-García, J. Tello, Photocatalytic 603 disinfection of natural well water contaminated by Fusarium solani using TiO2 slurry in solar CPC 604 photo-reactors, Catalysis Today, 144 (2009) 62-68. 605 32. OECD Guidelines for Testing of Chemicals, Simulation Test-Aerobic Sewage Treatment 606 303A, 1999. 607 33. M. Gibbs, A simple method for the rapid determination of iron in natural waters, Water 608 research, 13 (1979) 295-297. 609 18  34. F. Mendez-Arriaga, R.A. Torres-Palma, C. Petrier, S. Esplugas, J. Gimenez, C. Pulgarin, 610 Ultrasonic treatment of water contaminated with ibuprofen, Water research, 42 (2008) 4243-4248. 611 35. J. Marugán, R. van Grieken, C. Pablos, C. Sordo, Analogies and differences between 612 photocatalytic oxidation of chemicals and photocatalytic inactivation of microorganisms, Water 613 research, 44 (2010) 789-796. 614 36. P. Butz, B. Tauscher, Emerging technologies: chemical aspects, Food research international, 615 35 (2002) 279-284. 616 37. P. Fellows, Food processing technology: Principles and practice. (2000) 2nd ed., CRC Press, 617 New York 618 38. E.R. Holm, D.M. Stamper, R.A. Brizzolara, L. Barnes, N. Deamer, J.M. Burkholder, 619 Sonication of bacteria, phytoplankton and zooplankton: Application to treatment of ballast water, 620 Marine pollution bulletin, 56 (2008) 1201-1208. 621 39. T. Leighton, The acoustic bubble, Access Online via Elsevier, 1994. 622 40. A. Molina-Höppner, W. Doster, R.F. Vogel, M.G. Gänzle, Protective effect of sucrose and 623 sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments, Applied 624 and environmental microbiology, 70 (2004) 2013-2020. 625 41. C. Petrier et al, in: T.J. Mason, A. Tiehm, Advances in Sonochemistry, Volume 6: Ultrasound 626 in Environmental Protection, Access Online via Elsevier, 2001 627 42. R.S. Davidson, A. Safdar, J.D. Spencer, B. Robinson, Applications of ultrasound to organic 628 chemistry, Ultrasonics, 25 (1987) 35-39. 629 43. Y. Matsumura, A. Iwasawa, T. Kobayashi, T. Kamachi, T. Ozawa, M. Kohno, Detection of 630 High-frequency Ultrasound-induced Singlet Oxygen by the ESR Spin-trapping Method, Chemistry 631 Letters, 42 (2013) 1291-1293. 632 44. J. Ndounla, D. Spuhler, S. Kenfack, J. Wéthé, C. Pulgarin, Inactivation by solar photo-Fenton 633 in pet bottles of wild enteric bacteria of natural well water: Absence of re-growth after one week of 634 subsequent storage, Applied Catalysis B: Environmental, 129 (2013) 309-317. 635 45. F. Zaviska, P. Drogui, E.M. El Hachemi, E. Naffrechoux, Effect of nitrate ions on the 636 efficiency of sonophotochemical phenol degradation, Ultrasonics sonochemistry, 21 (2014) 69-75. 637 46. E. Novo, M. Parola, Redox mechanisms in hepatic chronic wound healing and fibrogenesis, 638 Fibrogenesis & tissue repair, 1 (2008) 5. 639 47. M.D. Kryszczuk, The effect of ultrasound in a chemotherapeutic study of ascites tumor cells, 640 in, Catholic University of America Press, Washington, 1962. 641 48. E. Dahl, Physicochemical aspects of disinfection of water by means of ultrasound and ozone, 642 Water research, 10 (1976) 677-684. 643 49. R.A. Torres, G. Sarantakos, E. Combet, C. Pétrier, C. Pulgarin, Sequential helio-photo-Fenton 644 and sonication processes for the treatment of bisphenol A, Journal of Photochemistry and 645 Photobiology A: Chemistry, 199 (2008) 197-203. 646 50. M. Chauhan, Effect of ultrasound on the redox reactions of iron (II) and (III), Indian journal of 647 chemistry. Sect. A: Inorganic, physical, theoretical & analytical, 43 (2004) 2098-2101. 648 51. Y.T. Didenko, S. Pugach, Spectra of water sonoluminescence, The Journal of Physical 649 Chemistry, 98 (1994) 9742-9749. 650 52. C. Petrier, A. Jeunet, J.L. Luche, G. Reverdy, Unexpected frequency effects on the rate of 651 oxidative processes induced by ultrasound, Journal of the American Chemical Society, 114 (1992) 652 3148-3150. 653 53. C. Petrier, M.-F. Lamy, A. Francony, A. Benahcene, B. David, V. Renaudin, N. Gondrexon, 654 Sonochemical degradation of phenol in dilute aqueous solutions: comparison of the reaction rates at 20 655 and 487 kHz, The Journal of Physical Chemistry, 98 (1994) 10514-10520. 656 54. C. Petrier, B. David, S. Laguian, Ultrasonic degradation at 20 kHz and 500 kHz of atrazine 657 and pentachlorophenol in aqueous solution: Preliminary results, Chemosphere, 32 (1996) 1709-1718. 658 19  55. S. Koda, M. Miyamoto, M. Toma, T. Matsuoka, M. Maebayashi, Inactivation of Escherichia 659 coli and Streptococcus mutans by ultrasound at 500kHz, Ultrasonics sonochemistry, 16 (2009) 655-660 659. 661 56. E. Joyce, A. Al‐Hashimi, T. Mason, Assessing the effect of different ultrasonic frequencies on 662 bacterial viability using flow cytometry, Journal of applied microbiology, 110 (2011) 862-870. 663 57. C.N. Haas, R.S. Engelbrecht, Physiological alterations of vegetative microorganisms resulting 664 from chlorination, Journal (Water Pollution Control Federation), (1980) 1976-1989. 665 58. S. Broekman, O. Pohlmann, E.S. Beardwood, E.C. de Meulenaer, Ultrasonic treatment for 666 microbiological control of water systems, Ultrasonics sonochemistry, 17 (2010) 1041-1048. 667 59. A. Hulsmans, K. Joris, N. Lambert, H. Rediers, P. Declerck, Y. Delaedt, F. Ollevier, S. Liers, 668 Evaluation of process parameters of ultrasonic treatment of bacterial suspensions in a pilot scale water 669 disinfection system, Ultrasonics sonochemistry, 17 (2010) 1004-1009. 670 60. A. Al Bsoul, J.P. Magnin, N. Commenges-Bernole, N. Gondrexon, J. Willison, C. Petrier, 671 Effectiveness of ultrasound for the destruction of Mycobacterium sp. strain (6PY1), Ultrasonics 672 sonochemistry, 17 (2010) 106-110. 673 61. E. Ortega-Gomez, P. Fernandez-Ibanez, M.M. Ballesteros Martin, M.I. Polo-Lopez, B. 674 Esteban Garcia, J.A. Sanchez Perez, Water disinfection using photo-Fenton: Effect of temperature on 675 Enterococcus faecalis survival, Water research, 46 (2012) 6154-6162. 676 62. S. Giannakis, E. Darakas, A. Escalas-Cañellas, C. Pulgarin, The antagonistic and synergistic 677 effects of temperature during solar disinfection of synthetic secondary effluent, Journal of 678 Photochemistry and Photobiology A: Chemistry, 280 (2014) 14-26. 679 63. A. Rincon, C. Pulgarin, Photocatalytical inactivation of E. coli: effect of (continuous–680 intermittent) light intensity and of (suspended–fixed) TiO2 concentration, Applied Catalysis B: 681 Environmental, 44 (2003) 263-284. 682 64. V. Raman, A. Abbas, Experimental investigations on ultrasound mediated particle breakage, 683 Ultrasonics sonochemistry, 15 (2008) 55-64. 684 65. E. Joyce, S.S. Phull, J.P. Lorimer, T.J. Mason, The development and evaluation of ultrasound 685 for the treatment of bacterial suspensions. A study of frequency, power and sonication time on 686 cultured Bacillus species, Ultrasonics sonochemistry, 10 (2003) 315-318. 687 66. N. Klamerth, S. Malato, A. Agüera, A. Fernández-Alba, G. Mailhot, Treatment of Municipal 688 Wastewater Treatment Plant Effluents with Modified Photo-Fenton As a Tertiary Treatment for the 689 Degradation of Micro Pollutants and Disinfection, Environmental Science & Technology, 46 (2012) 690 2885-2892. 691 67. J. Rodríguez-Chueca, M.I. Polo-López, R. Mosteo, M.P. Ormad, P. Fernández-Ibáñez, 692 Disinfection of real and simulated urban wastewater effluents using a mild solar photo-Fenton, 693 Applied Catalysis B: Environmental, 150–151 (2014) 619-629. 694 695 20  List of Tables 696 Table 1 – Hydraulic characteristics of previous works in CPC reactors 697 Flow rate (L/min) Total volume (L) Volume-toflowrate ratio Illuminated Volume Volume in the dark Light-todark ratio FernandezIbañez et al. (2009) 20 14 0.70 32% 68% 0.47 MoncayoLasso et al. (2009) 17.5 20 1.14 45% 55% 0.82 FernandezIbañez et al. (2005) (varied flowrates) 5, 13, 22.5 11 2.2, 0.85, 0.49 49% 51% 0.96 Rincon & Pulgarin (2007) (min, max capacity) 20.5 37, 70 1.80, 3.41 65%, 34% 35%, 66% 1.86, 0.52 Sciacca et al. (2011) 24.2 18 0.74 83% 17% 4.88 Giannakis et al. (2013) 0.03, 0.06, 0.07 0.7 22.58, 12.28, 9.59 33% 67% 0.49 Ndounla et al. (2013) 2 25 12.5 60% 40% 1.53 698 Table 2 – Parameters involved in the joint treatment process 699 Factors Values Other parameters Light 1000 W/m2Temperature: 30˚C Ultrasound 20 W Recirculating Flow rate: 4.39 L/h Iron 1 ppm Treated Volume: 500 mL H2O2 10 ppm Initial Population: 106 CFU/mL 700 701 21  Table 3 – Subsets of experiments in the step-wise construction of the joint hv/US/Fe/H2O2 702 treatment process. 703 Experiments Treatment constituents 1-2 WW and WW/Fe/H2O2 ‐ Wastewater with no treatment ‐ Wastewater + Fe/H2O2 3-4 US and US/Fe/H2O2 ‐ Wastewater+US ‐ Wastewater+US+Fe/H2O2 5-6 hv and hv/Fe/H2O2 ‐ Light ‐ Light+/Fe/H2O2 (photo-Fenton) 7-8 hv/US and hv/US/Fe/H2O2 ‐ Light+US ‐ US+photo-Fenton 704 Table 4 – Overview of the investigation of the operational parameters 705 Factors1 Level 1 Level 2 Level 3 Hydraulic Pump rpm 33 66 99 No. of Illuminated vessels 1 2 3 Wastewater volume (mL) 500 600 700 Environmental Temperature (˚C) 10 20 30 Light Intensity (W/m2) 800 1000 1200 Fenton / Ultrasound H2O2 Concentration (ppm) 5 10 20 Fe Concentration (ppm) 0.5 1 2 US Acoustic Power (W) 10 20 40 1Central values are annotated with bold. 706 707 22  708 Table 5 – Hydraulic calculations on the reactor set-up 709 710 Increasing recirculation speed from 33 to 99 rpm (1.87 to 4.39 L/h) Reactors333rpmReactors366rpmReactors399rpm 23  711 Volume500mL1.87L/h1.87L/hVolume500mL3.44L/h3.44L/hVolume500mL4.39L/h4.39L/h Light230mL7.38min46%Light230mL4.01min46%Light230mL3.14min46% Tubing70mL2.25min14%Tubing70mL1.22min14%Tubing70mL0.96min14% US200mL6.42min40%US200mL3.49min40%US200mL2.73min40% Total500mL16.04min100%Total500mL8.72min100%Total500mL6.83min100% Increasing illuminated volume from 1 reactor to 3 (75 to 230 mL) Reactors199rpmReactors299rpmReactors399rpm Volume500mL4.39L/h4.39L/hVolume500mL4.39L/h4.39L/hVolume500mL4.39L/h4.39L/h Light75mL1.03min15%Light150mL2.05min30%Light230mL3.14min46% Tubing50mL0.68min10%Tubing60mL0.82min12%Tubing70mL0.96min14% US375mL5.13min75%US290mL3.96min58%US200mL2.73min40% Total500mL6.83min100%Total500mL6.83min100%Total500mLmL6.83min100% Increasing total treated volume from 500 to 700 mL Reactors399rpmReactors399rpmReactors399rpm Volume500mL4.39L/h4.39L/hVolume600mL4.39L/h4.39L/hVolume700mL4.39L/h4.39L/h Light230mL3.14min46%Light230mL3.14min38%Light230mL3.14min33% Tubing70mL0.96min14%Tubing70mL0.96min12%Tubing70mL0.96min10% US200mL2.73min40%US300mL4.10min50%US400mL5.47min57% Total500mL6.83min100%Total600mL8.20min100%Total700mL9.57min100% 24  List of Figures 712 713 Figure 1 – Experimental set-up, consisting of the illuminated area (Suntest apparatus), the recirculation pump, 714 the (dark) sonication vessel and the temperature control (thermostat). The flow direction is clockwise, water is 715 introduced at surface level and sampled from the bottom of the vessel. 716 717 718 Figure 2 – Experimental results from the coupling of photo-Fenton and sonication. a) Experiments 1-2 (WW and 719 WW/Fe/H2O2), b) Experiments 3-4 (US and US/Fe/H2O2), c) Experiments 5-6 (hv and hv/Fe/H2O2) and d) 720 Experiments 7-8 (hv/US and hv/US/Fe/H2O2). e) Long-term inactivation events for 48 h (time axis initiates in the 721 4-h mark, after treatment). 722 723 25  724 Figure 3 – Suggestion of the added actions sonication has towards bacterial inactivation, when coupled with 725 photo-Fenton. The known photo-Fenton mechanisms suggested by literature are not displayed. 726 727