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

1  Ul asound enhancemen o nea -neu al pho o-Fen on o e ec i e E. coli 1 inac i a ion in was ewa e 2 3 S e anos Giannakis1,2,3, S e anos Papou sakis3, E hymios Da akas1, An oni Escalas-4 Cañellas2,4, Ch is ian Pé ie 5,6, Césa Pulga in3,* 5 1Labo a o y o En i onmen al Enginee ing and Planning, Depa men o Ci il Enginee ing, A is o le Uni e si y o 6 Thessaloniki, 54624 Thessaloniki, G eece 7 2Labo a o y o Con ol o En i onmen al Con amina ion, Ins i u e o Tex ile Resea ch and Indus ial Coope a ion o Te assa 8 (INTEXTER), Uni e si a Poli ècnica de Ca alunya, Colom 15, 08222 Te assa, Ca alonia, Spain 9 3Swiss Fede al Ins i u e o Technology, Lausanne, Ins i u e o Chemical Sciences and Enginee ing, 1015 Lausanne, 10 Swi ze land 11 4Depa men o Chemical Enginee ing & Te assa School o Enginee ing, Uni e si a Poli ècnica de Ca alunya, Colom 1, 12 08222, Te assa, Ca alonia, Spain 13 5Labo a oi e Rhéologie e P océdés, G enoble-INP, UMR CNRS 5520, BP 53, 38041 G enoble Cedex 9, F ance 14 6King AbdulAziz Uni e si y, Jeddah, Saudi A abia 15 *Co esponding au ho : Césa Pulga in, Tel: +41216934720; Fax: +41216936161; E-mail: 16 [email p o ec ed] 17 18 Abs ac 19 In his s udy, we a emp o he i s ime o couple sonica ion and pho o-Fen on o bac e ial 20 inac i a ion o seconda y ea ed e luen . Syn he ic was ewa e was subjec ed o sequen ial high-21 equency/low powe sonica ion, ollowed by mild pho o-Fen on ea men , unde a sola simula o . I 22 was ollowed by he assessmen o he con ibu ion o each componen o he p ocess (Fen on, US, h ) 23 owa ds he emo al a e and he long- e m su i al; sunligh g ea ly imp o ed he ea men 24 e iciency, wi h he coupled p ocess being he only one o yield o al inac i a ion wi hin he 4-h pe iod 25 o ea men . The sho - e m bene icial disin ec ing ac ion o US and i s de imen al e ec on bac e ial 26 su i al in long e m, as well as he impac o ligh addi ion we e also e ealed. Finally, an 27 in es iga ion on he ope a ional pa ame e s o he p ocess was pe o med, o in es iga e possible 28 imp o emen and/o limi a ions o he coupled ea men ; 3 le els o each pa ame e in ol ed 29 (hyd aulic, en i onmen al, US and Fen on) we e es ed. Only H2O2 inc eased imp o ed he p ocess 30 signi ican ly, bu he ac ion mode o he join p ocess indica ed po en ial cos -e ec i e solu ions 31 owa ds he implemen a ion o his me hod. 32 Keywo ds: was ewa e disin ec ion, pho o-Fen on, high- equency ul asound, E. coli, inac i a ion 33 mechanism34 NOTICE: hisis heau ho ’s e siono awo k ha wasaccep ed o publica ioninUl asonicsSonochemis y .Changes esul ing om hepublishingp ocess,suchas pee  e iew,edi ing,co ec ions,s uc u al o ma ing,ando he quali ycon olmechanismsmayno be e lec edin hisdocumen .Changesmayha ebeenmade o hiswo ksincei wassubmi ed o publica ion.Ade ini i e e sionwassubsequen lypublishedinUl asonicsSonochemis y[Vol.22,p.515‐526,Janua y2015].DOI: 10.1016/j.ul sonch.2014.04.015 2  1. INTRODUCTION 35 36 Ad anced Oxida ion P ocesses (AOPs) ha e been in he spo ligh o mo e han h ee decades, as pa 37 o a global e o o mode nize ac ual me hods o wa e disin ec ion. Thei ac ion is based on he 38 p oduc ion o he ex emely oxidizing hyd oxyl adical (•OH) [1], which can a ack he chemical 39 s uc u e o he mic oo ganisms’ cell wall and inac i a e hem [2]. Ul asound has been ex ensi ely 40 s udied as an AOP, a ge ing mic oo ganism inac i a ion, such as bac e ia, i uses e c., by ei he low 41 (~20 kHz) o high equencies (200+ kHz) [2, 3, 4 and 5]. This me hod is exploi ing he di ec 42 mechanical ac ion o he ca i a ion bubble implosion (low equencies) as well as he addi ional 43 p oduc ion o H2O2 and •OH adicals du ing ca i a ion (high equencies); he p opaga ion o 44 ul asound wa es in he aqueous medium ini ia es he a o emen ioned ac ions, by he gene a ion o 45 ex eme empe a u e and p essu e condi ions [6], which ha e a p o en bac e icidal e ec [7, 8, 9, and 46 10]. 47 As a as he ul asound se -up is conce ned, he equency o he ul asonic wa es is a c ucial 48 pa ame e , o i de ines he size o he ca i a ion bubbles [11]. Li e a u e sugges s ha he a e age 49 ca i y size is p opo ional o he acous ic powe and in e sely p opo ional o he ul asound equency 50 [12]. I is also e i ied ha apa om low equency/high powe ul asound sys ems [13, 14], high 51 equency/low powe p ocesses ha e been p o en o e icien ly inac i a e mic oo ganisms [8, 15, and 52 16]. Howe e , ul asound al eady equi es high in ensi ies o achie e o al inac i a ion o 53 mic oo ganisms, and he e o e, is conside ed an expensi e applica ion o la ge olumes o wa e [2]. 54 Conside ing all he abo e, i should be used p e e ably as a complemen a y disin ec ing me hod [2]. 55 The pho o-Fen on p ocess [17] could play he ole o he main disin ec ing me hod, as one o he mos 56 e icien me hods o hyd oxyl adical p oduc ion [18]. La ely, i has e en been used o disin ec 57 d inking wa e , being a good al e na i e o chlo ina ion, wi h i s known disin ec ion by-p oduc s 58 o ma ion [19]. Howe e , was ewa e is a complex ma ix in which many o ganic and ino ganic 59 compounds coexis , such as nu ien s, sal s and many subs ances ha could in luence he ou come o 60 he applica ion o ei he p ocess. I has been epo ed ha he p esence o hyd oxyl adical sca enge s, 61 namely he o ganic ma e , p esen s an addi ional oxida ion a ge and ende s AOPs sensi i e o he 62 ea men o was ewa e [20, 21]. Supp ession o hese sca enge s e ealed hei impo ance [10] and 63 also, o yea s he Fen on eac ion was belie ed o be a pH- es ic ed eac ion in highly acidic egions; 64 i was conside ed impossible o apply such me hods, in ma ices wi h nea -neu al pH [22]. Howe e , 65 ecen ad ances [18, 23 and 24] ha e p o en i s e ec i eness in he neu al a ea, and in he 66 simul aneous p esence o o ganic ma e [18, 25]. P e ious wo k in ou g oup has shown, he e is no 67 need o acidi ica ion p io o he ea men o keep a signi ican pa o he i on soluble; apa om he 68 di ec complexion wi h bac e ia, he e a e some s ong pho oac i e Fe3+ complexes o med in p esence 69 o o ganic ma e [21, 25]: 70 3  [R-COO- - Fe3+]2+  h  Fe2+ + CO2 + R• (1) 71 The cycle con inues wi h he eac ion o he egene a ed i on wi h hyd ogen pe oxide o p oduce mo e 72 hyd oxyl adicals e c. 73 Fe2+ + H2O2  OH- + •OH + Fe3+ (2) 74 In o de o inc ease he amoun o wa e ea ed by sola -assis ed me hods, compound pa abolic 75 collec o eac o s ha e been used [17, 18, 23 and 26], and sola pho o-Fen on e en was a subjec unde 76 ques ion, because o he in e mi en ac ion o he ligh [27]. The e is a echnical issue o be add essed 77 in he in e mi en na u e o his ea men me hod, and he exis ence o “dead” ime among he 78 expe imen . Typically, a CPC pho o- eac o consis s o he illumina ed su ace and he s o age-79 eci cula ion ank. The eci cula ing low o hese eac o s c ea es a gap in he illumina ion o as long 80 as wa e is p esen in he (da k) s o age ank, allowing bac e ial de ense mechanisms o deploy [28]. 81 Li e a u e indica es a a ie y o ligh - o-da k dis ibu ions (Table 1), which ma e ialize his di e ence 82 [17, 18, 23, 26, 29, 30 and 31]. 83 The e o e, keeping in mind he imp o emen o he nea -neu al pho o-Fen on disin ec ion while 84 wo king wi hin ealis ic ope a ional pa ame e s, o he i s ime we s udy he join ul asound/pho o-85 Fen on ea men o was ewa e , in a CPC-like, lab-scale sys em. In his manne , we will ake 86 ad an age o wo ac o s ha could wo k complemen ing each o he : i s ly, he exploi a ion o he 87 da k in e als o sonica ion, along wi h he u iliza ion o sola ene gy o he p omo ion o a mild 88 pho o-Fen on eac ion and secondly, he supplemen a y ac ion hese p ocesses ha e, since, o 89 ins ance, US can p oduce H2O2 and subsequen ly, could uel he pho o-Fen on p ocess. In ou s udy, 90 syn he ic seconda y e luen was used, spiked wi h E. coli K12, eci cula ing a ound a sonica ed da k 91 eac o and an illumina ed ba ch eac o , unde sola simula ed ligh . We aim o: 92 i) Explo e he e ec s o he pho o-Fen on ac o s (ligh , eac an s) and he ul asonic ac ion 93 (US) on bo h sho and long- e m disin ec ion e en s; cla i ica ion o he e ec s is 94 a emp ed by s epwise inse ion o he pa icipa ing ac ions. 95 ii) In es iga e he in ol ed ope a ional pa ame e s ( eci cula ion speed, empe a u e, ligh 96 in ensi y, ea ed olume and dis ibu ion o olumes, i on and hyd ogen pe oxide con en , 97 ul asound in ensi y) in a small-scale se -up. 98 99 2. MATERIALS AND METHODS 100 101 2.1. Syn he ic seconda y e luen p epa a ion 102 103 4  2.1.1. Mic obial me hods 104 The E. coli s ain K12 (MG1655) employed was p o ided by he “Deu sche Sammlung on 105 Mik oo ganismen und Zellkul u en”. Lu ia-Be ani b o h was inocula ed wi h a colony om bac e ial 106 E. coli p e-cul u es, placed in 50 ml plas ic alcons o 8 h and hen loop inocula ed, a e 1% dilu ion 107 o e nigh (180 pm and 37°C o 15 h), o achie e s a iona y phase cells. 108 Ha es ed cells we e cen i uged and washed h ee imes (5000 pm, 15 and 5 min o sepa a ion and 109 washing, espec i ely), ollowed by ese a ion in saline solu ion (neu al pH solu ion wi h 8 g/L NaCl 110 and 0.8 g/L KCl); a solu ion o 109 CFU/mL is achie ed. 111 112 2.1.2. Syn he ic was ewa e composi ion 113 The p epa a ion o he syn he ic was ewa e ook place as unde he di ec i e o SYMAWE [32]. The 114 ini ial DOC was 100 mg/L (250 mg/L COD). The expe imen s used a 10% dilu ion (in dis illed wa e ) 115 o he said composi ion. The dilu ion pe o med co esponds o he COD and DOC alues encoun e ed 116 in no mal seconda y e luen s. Finally, he pH o he sample was be ween 6.5-7. 1 mL o he p epa ed 117 bac e ial solu ion was used o spike he dilu ed was ewa e , hus esul ing in an ini ial bac e ial 118 popula ion o 106 CFU/mL. 119 120 2.2. Reagen s and analyses 121 122 The was ewa e cons i uen s, as well as he Fen on eagen s we e used as ecei ed. Pho o-Fen on 123 expe imen s we e ca ied ou employing e ous sul a e hep ahyd a e (Fluka Chemika), hyd ogen 124 pe oxide (35% by weigh , Sigma Ald ich), used as ecei ed. The dissol ed i on (Fe2+, Fe3+) was 125 measu ed wi h he e ozine me hod [33], using a UV-Vis Lambda 20 spec opho ome e , p o ided by 126 Pe kinElme , Schwe zenbach, Swi ze land. Fo 1.6 mL o sample 0.2 o e ozine solu ion (4.9 mM) 127 was added, ollowed by 0.2 mL o hyd oxylamine hyd ochlo ide solu ion 10% w/w. Ace a e bu e 128 solu ion was added o a inal 4.5-5 pH alue. To de e mine he concen a ion o hyd ogen pe oxide in 129 he sample i anium oxysul a e solu ion was added, also measu ed wi h he same spec opho ome e . 130 The pH o each sample was measu ed wi h a pH-me e p o ided by Me le Toledo (PH/Ion S220, 131 Se en Compac , Me le Toledo). 132 133 2.3. Desc ip ion o eac o s’ se -up 134 135 The p elimina y s udy was ca ied ou in plain Py ex glass ba ch eac o s o 65 mL o al capaci y. In 136 he se -up p esen ed in Figu e 1, he con igu a ion pe mi s he sequen ial ea men o he syn he ic 137 5  was ewa e ; US/pho o-Fen on ea men was aking place (o ice e sa). The same con igu a ion was 138 used in one o ou p e ious wo ks, (used in [29], simila o he se -up used by Mendez-A iaga e al, 139 2009 [34]), syn he ic was ewa e om a cylind ical double-wall glass essel (400 mL) was pumped by 140 a pe is al ic pump h ough h ee glass eac o s (diame e 3.8 cm, e ec i e i adia ion su ace 214.8 141 cm2), connec ed in se ies, o o al olume 230 mL. Tempe a u e was egula ed by wa e eci cula ing 142 a ound he eac o and connec ed o a he mos a . The hi d eac o e luen was eci cula ed o he 143 o iginal essel. No mally, wa e was inse ed in he double-wall eac o and pumped in o he i adia ed 144 pa . The e o e, 230 mL o wa e we e always p esen unde illumina ion, 70 mL in he dis ibu ion 145 sys em and he es subjec ed o sonica ion. 146 The ul asonic wa es (275 kHz) we e emi ed om a piezoelec ic 4-cm disc, ixed on a Py ex glass 147 pla e adjus ed o he bo om o he double-walled eac o . The in ensi ies applied in all expe imen s 148 we e 10, 20 and 40 W. The elec ic powe was he chosen me hod o calib a e he ul asonic 149 equipmen . The in-se ies eac o s we e i adia ed by he Sun es appa a us. The Sun es CPS sola ligh 150 simula o bea s a lamp ha emi s ~0.5% o he pho ons a wa eleng hs <300 nm, ~7% be ween 300 151 and 400 nm and he es ollow he sola spec um. The global i adiance alues used in his wo k we e 152 800, 1000 and 1200 W/m2, while he co esponding UV alues we e app oxima ely 19.2, 24.7 153 and 30.2 W/m2. 154 155 2.4. Expe imen al design 156 157 Two se s o expe imen s we e pe o med. In a i s se o 8 expe imen s, ha we call s ep-wise 158 cons uc ion o he join ea men p ocess, he elemen s o he US/h /Fe/H2O2 we e g adually and 159 accumula i ely applied o he was ewa e , in o de o de e mine he indi idual ole o each ac o and 160 o de ec any syne gy among hem. Table 2 shows he condi ions co esponding o each indi idual 161 ea men ac o when applied. Table 3 summa izes he ou subse s o expe imen s in he s ep-wise 162 design. 163 In a second se o expe imen s (imp o emen o he p ocess e iciency), eigh di e en a iables we e 164 indi idually modi ied a h ee le els, while keeping he o he a iables cons an , in o de o ob ain 165 imp o ed wo king le els o each a iable. Table 4 displays he h ee alues (le els) essayed o each 166 a iable. In each expe imen he emaining pa ame e s we e kep cons an and se o he cen al alue 167 shown in he able. 168 169 2.5. Bac e ial enume a ion and eg ow h es s 170 171 6  The disin ec ion e iciency was measu ed by iable pla e coun s on Pe i dishes con aining PCA aga 172 (plas ic, 9-cm diame e ). The pou -pla ing me hod was used and dilu ions we e made o ensu e 173 coun able numbe s on he pla es, i.e. 20-100 colonies/pla e. Expe imen s we e pe o med wice and 174 pla ing ook place in 2-3 consequen dilu ions and in duplica es. 175 Reg ow h o bac e ia was es ima ed a e he s o age o he samples a ambien empe a u e o 24 and 176 48 h a e he sampling ime. Samples we e kep in 1.5 mL plas ic Eppendo caps in he da k and he 177 popula ion was measu ed o assess he pos -i adia ion e en s, a e hei emo al om he 178 expe imen al se -up. 179 180 3. RESULTS AND DISCUSSION 181 182 3.1. Resul s o he s ep-wise cons uc ion o he join ea men p ocess 183 184 As a as a po en ial applica ion o mild pho o-Fen on assis ed by high equency/low powe 185 ul asound is conce ned, mode a e concen a ions o eac an s a e sugges ed o he e olu ion o ou 186 s udy, a e an ini ial in es iga ion (da a no shown). A 1000 W/m2 ligh in ensi y, an addi ion o 1 187 ppm i on and 10 ppm o H2O2 will be used, as ma ginal alues o Fen on eagen s and 20 W o US 188 powe . 189 190 3.1.1. Disin ec ion e iciency 191 192 i) Expe imen s: 1-2 (WW and WW/Fe/H2O2). 193 Figu e 2a p esen s he esul s o he i s pa o he expe imen s, whe e nei he ligh no US was 194 applied. Was ewa e was eci cula ed a ound he non-illumina ed, non-sonica ed expe imen al se -up 195 and he co esponding g aphs desc ibe he changes when H2O2 and i on we e added o he solu ion. 196 We no ice he inc ease o he popula ion, when no eac an s we e added, due o he exis ence o 197 nu ien s and sal s ha a o bac e ial g ow h in his wa e ma ix [35]. H2O2 is a subs ance wi h 198 disin ec ing ac ion, while i on i sel is no oxic o bac e ia. The addi ion o bo h eac an s causes he 199 ini ia ion o he Fen on eac ion, which has a slow, bu exis ing disin ec ing abili y and wi hin a 200 ime ame o 4 h, we obse e a 24.4% educ ion in he ini ial popula ion. 201 202 ii) Expe imen s: 3-4 (US and US/Fe/H2O2). 203 7  Figu e 2b demons a es he e ec s sonica ion has on samples, alongside wi h he s epwise inse ion o 204 he Fen on eagen s. The sample eci cula es a ound he ul asound essel and he non-illumina ed 205 a ea, being subjec o in e mi en high- equency, low in ensi y sonica ion. When ul asound alone is 206 applied, he e is a dec ease in o al bac e ial numbe s, app oaching 27.9%. The concu en addi ion o 207 bo h Fen on eac an s (H2O2 and Fe2+) in he sonica ed sample causes an 82.1% educ ion in he 208 bac e ial popula ion, compa ed o 27.9 % educ ion o US ea men and 24.4% o Fen on ea men 209 alone. This indica es a syne gy be ween sonica ion and he Fen on eagen s; a syne gy ac o o 1.57 is 210 demons a ed by he disin ec ing e iciency o he eac ions. 211 Du ing sonica ion, he b eakage o he ca i a ion bubbles can lead o he o ma ion o an almos poin -212 sized hea sou ce [36, 37], wi h local empe a u es app oaching 2000 K and p essu es o 200 a m. 213 These ex eme condi ions can cause lysis o wa e molecules and along wi h ha , ex a p oduc ion o 214 hyd oxyl adicals [8]. The p esence o he a o e-men ioned pa icles in eal was ewa e and he 215 bac e ia (in ou ma ix) could also play ano he impo an ole, since he collapse o he ca i a ion 216 bubbles nea a pa icle in he medium could cause mic o-je s, depending on he size o he pa icle [38] 217 and could also o m “weak spo s” in he body o he liquid; hese a e po en ial places o o m a ca i y 218 [39]. I has been also epo ed ha he p esence o some sal s causes a ba o-p o ec i e e ec on he 219 cells [40] and samples wi h highe con en s o soluble solids would equi e highe sonica ion imes. 220 Apa om he physical damage, du ing he ul asound ea men o he sample, he e is ample 221 gene a ion o eac i e oxygen species (•OH adicals [41], single oxygen [42, 43]), as men ioned 222 be o e, which a e known o s ess bac e ia and lead o cell dea h [25, 44]. 223 Finally, he addi ion o pep one (p esen in he syn he ic was ewa e ) and he gene ally, p esence o 224 ni ogen compounds has been epo ed o delay he sonica ed deg ada ion o phenols [45]. Howe e , 225 ni ogen, unde he p esence o ul asound wa es can o m NOx (ni a e and ni i e). I s eac ion wi h 226 single oxygen (as p oduced be o e) [43] p oduces pe oxyni i e (ONOO-) [46]. Pe oxyni i e is 227 included in he eac i e ni ogen species and can cause signi ican inju es o a ious s uc u es o he 228 cell ( ee adical damage o a ack agains he espi a o y chain) [46]. 229 The syne gis ic ac ion o US and Fen on p ocesses can be a ibu ed o he exploi a ion o he 230 ecombined H2O2 ( om •OH), which is less oxida i e han he hyd oxyl adical i sel , and wi h ha , he 231 e-ini ia ion o he Fen on eac ion wi h new eac an s. Also, he ul asound p ocess, acco ding o 232 K yszczuk [47], inc eases he ansien b eakage o he bonds among he molecula componen s o he 233 cell memb ane, which inc eases he pe meabili y o he cell in ex e nal subs ances [48]. The e o e, he 234 in oduc ion o Fe2+ in he cell is easie and i s p esence inside he cell can p oduce hyd oxyl adicals 235 e y close o i al unc ions o he cell, as well as he DNA [25] due o he induced in e nal Fen on 236 p ocess. 237 238 iii) Expe imen s: 5-6 (h and h /Fe/H2O2). 239 8  The 3 d se o expe imen s is dedica ed in he in es iga ion o he impac o ligh in he sequen ial 240 p ocess. In all expe imen s ligh is p o ided a 1000 W/m2, bu in o al, in e mi en i adia ion is 241 p o ided o he sys em; he e is an illumina ed egime and a non-illumina ed one, in he Sun es 242 appa a us and he (inac i e) sonica ion essel (and ubing). In one o ou p e ious wo ks [29], we 243 demons a ed he impac ligh in e mi ence has on bac e ial disin ec ion and su i al, while 244 con inuous supply o e y as eci cula ion a ound illumina ed and da k egimes a o s disin ec ion, 245 wi h he same se -up. The e o e, pho o-Fen on is p omo ed in non-in e mi en egimes o , as in ou 246 case, sho da k in e al pe iods. 247 As i can be seen om Figu e 2c, ligh , e en in non-con inuous o m, is e y e ec i e and esul s in 248 high inac i a ion a es. I s disin ec ing ac ion is domina ing he emo al p ocess, un il he Fen on 249 eagen s a e p esen , and sola -assis ed pho o-Fen on is induced. The ac ion o pho o-Fen on is aking 250 place wi hin he Sun es and da k (no mal) Fen on akes place du ing he es o he ime, in a 0.85:1 251 ime dis ibu ion (46% pho o-Fen on o e 54% Fen on). A e an ini ial delay, which is demons a ed 252 as a shoulde in he g aph, eac ion is mo e e ec i e by he h /Fe/H2O2 han he co esponding sola 253 ea men . 254 Spuhle e al. [25] ha e e iewed he mechanism o bac e ial inac i a ion by he pho o-Fen on eac ion 255 in nea -neu al wa e wi h o ganic componen s, and ha e sugges ed he possible sou ces o ROS 256 p oduc ion and cellula pho o-oxida i e damage, as well as he damage done by he ROS hemsel es, 257 de i ing om he pho o-Fen on eac ion. In ou sugges ed ea men me hod, hese mechanisms a e 258 comple ely compa ible, explaining he majo i y o he ac ions and o he wo ks on nea -neu al pho o-259 Fen on mechanisms desc ibe ully he mechanisms, so we will no u he analyze hei indings. 260 261 i ) Expe imen s: 7-8 (h /US and h /US/Fe/H2O2). 262 The inal g oup o expe imen s a e p esen ed in Figu e 2d. This g aph summa izes he esul s o he 263 join ea men by ligh and ul asound. I is clea , a e a compa ison wi h Figu e 2c, ha when ligh is 264 p esen , i s disin ec ing ac ion is domina ing he p ocess. Howe e , we obse e ha he only case o al 265 disin ec ion is achie ed, is by he sequen ial US/pF sys em. In his sys em, was ewa e spends i s ime 266 dis ibu ed 46% unde pho o-Fen on, 14% in he da k (da k-Fen on) and 40% in he sonica ion essel 267 (US/Fen on). The expe imen al ime has less inac i e pe iods, and we obse e ha i has a signi ican 268 impac in he o al inac i a ion o he bac e ial popula ions in less han 4h. He e, he pho o-Fen on/US 269 syne gy is low in e ms o bac e ial coun s, e iciency was imp o ed in a ela i ely low pe cen age, bu 270 only he coupled p ocess esul ed in o al disin ec ion in 4 h. The ele a ed e iciency and o al 271 inac i a ion o he i s ime, is a ibu ed o he combina ion o all he p e ious ac ions (in US and/o 272 ligh ), as well as he ollowing ac ions (a g aphical summa y o all he ac ions is p esen ed in Figu e 273 3): 274 9  i. The hyd oxyl adical is a sho li ing ROS, and i occu s no o each he a ge in all cases and 275 o en ecombines o c ea e H2O2 [49]. The e o e, he addi ion o ul asound di ec ly p oduces 276 hyd oxyl adicals and H2O2; he •OH di ec ly a acks he cell and H2O2 pa icipa es in he 277 pho o-Fen on eac ion (2). Alongside wi h he added H2O2, he e is addi ional p oduc ion, 278 ueling he Fen on eac ion and hus, imp o ing he o e all e iciency o he ea men . 279 ii. As we desc ibed be o e, wi h ul asound wa es, he loosening o ansien bonds and inse ion 280 o Fe2+ in he cell is inc eased, which p omo es he in e nal Fen on eac ion. A e he 281 comple ion o he Fen on eac ion, ligh educes Fe3+ o Fe2+, and e-ini ia es a adical 282 p oduc ion inside he cell (in e nal pho o-Fen on). 283 iii. Low equency ul asound has been p o en [50] o educe Fe3+ in he o m o e ous ions 284 (Fe2+). The a e age size o he bubble howe e dec eases when equency is inc eased, in ou 285 sys em [12]; ne e heless, ca i a ion s ill akes place. The e o e, i is possible ha an ac ion 286 like his could p o ide an addi ional sou ce o i on a ailable o he pho o-Fen on p ocess, and 287 p og ess he egene a ion o he ca alys in he (o he wise) non-illumina ed pa o he ime. In 288 ha way, mo e a ailable e ous ions can be p esen in he solu ion. 289 i . We men ioned he ex eme empe a u e and p essu e condi ions ha ake place du ing he 290 collapse o he ca i a ion bubbles. The in e io pa o he bubble, unde hese condi ions, is 291 known o emi ligh , unde he phenomenon o sono-luminescence [8]. The op ical aspec s o 292 his phenomenon ha e been s udied [51] and he emi ed ligh wa eleng hs all in o he 293 necessa y ones possibly able i) o induce he egene a ion o he pho o-Fen on eac ion 294 ca alys , ii) in lic di ec UV damage o he cell. Howe e , he necessa y ene gy o achie e his 295 is s ill unde ques ion. 296 . Apa om he adicals’ p oduc ion h ough he no mal pho o-Fen on cycle, he p esence o 297 ligh is pa icipa ing in ano he se ies o eac ions wi h ni ogen compounds. The pho olysis o 298 ni a e and ni i es (p oduced by he pa icipa ion o he US in he p ocess) can lead o 299 addi ional hyd oxyl adical p oduc ion [45]: 300 Pho olysis o Ni a e: 301  →    (3) 302  →      (4) 303    ↔    (5) 304 Pho olysis o Ni i e: 305  →     (6) 306    ↔    (7) 307 308 16  Swiss Go e nmen o he Swiss Go e nmen Excellence Schola ship, by means o which S e anos 518 Giannakis has ecei ed a Resea ch Visi ellowship (No. 2012.0499). Finally, S e anos Papou sakis 519 was unded by he Swiss-Hunga ian Co-ope a ion P og am “Sus ainable ine chemical, 520 pha maceu ical indus y: sc eening and u iliza ion o liquid was es – Inno a i e app oaches o he 521 aba emen o indus ial/ oxic was e in aqueous e luen s”. 522 523 6. REFERENCES 524 525 1. I. Olle , S. Mala o, J.A. Sánchez-Pé ez, W. Ge njak, M.I. Maldonado, L.A. Pé ez-Es ada, C. 526 Pulga ín, A combined sola pho oca aly ic-biological ield sys em o he mine aliza ion o an 527 indus ial pollu an a pilo scale, Ca alysis Today, 122 (2007) 150-159. 528 2. A. An oniadis, I. Poulios, E. Nikolakaki, D. Man za inos, Sonochemical disin ec ion o 529 municipal was ewa e , Jou nal o haza dous ma e ials, 146 (2007) 492-495. 530 3. P. Folado i, B. Lau a, A. Gianni, Z. Giuliano, E ec s o sonica ion on bac e ia iabili y in 531 was ewa e ea men plan s e alua ed by low cy ome y-- ecal indica o s, was ewa e and ac i a ed 532 sludge, Wa e esea ch, 41 (2007) 235-243. 533 4. C.V. Ch ysikopoulos, I.D. Mana io is, V.I. Syngouna, Vi us inac i a ion by high equency 534 ul asound in combina ion wi h isible ligh , Colloids and su aces. B, Bioin e aces, 107 (2013) 174-535 179. 536 5. T.J. Mason, E. Joyce, S.S. Phull, J.P. Lo ime , Po en ial uses o ul asound in he biological 537 decon amina ion o wa e , Ul asonics sonochemis y, 10 (2003) 319-323. 538 6. Y.T. Didenko, W.B. McNama a, K.S. Suslick, Ho spo condi ions du ing ca i a ion in wa e , 539 Jou nal o he Ame ican Chemical Socie y, 121 (1999) 5817-5818.. 540 7. I. Hua, M.R. Ho mann, Op imiza ion o ul asonic i adia ion as an ad anced oxida ion 541 echnology, En i onmen al Science & Technology, 31 (1997) 2237-2243. 542 8. I. Hua, J.E. Thompson, Inac i a ion o Esche ichia coli by sonica ion a disc e e ul asonic 543 equencies, Wa e esea ch, 34 (2000) 3888-3893. 544 9. M. Fu u a, M. Yamaguchi, T. Tsukamo o, B. Yim, C. S a a ache, K. Hasiba, Y. Maeda, 545 Inac i a ion o Esche ichia coli by ul asonic i adia ion, Ul asonics sonochemis y, 11 (2004) 57-60. 546 10. M.F. Dadjou , C. Ogino, S. Ma sumu a, N. Shimizu, Kine ics o disin ec ion o Esche ichia 547 coli by ca aly ic ul asonic i adia ion wi h TiO2, Biochemical Enginee ing Jou nal, 25 (2005) 243-548 248. 549 11. K.S. Suslick, Sonochemis y, Science, 247 (1990) 1439-1445. 550 12. A. B o chie, F. G iese , M. Ashokkuma , E ec o powe and equency on bubble-size 551 dis ibu ions in acous ic ca i a ion, Physical e iew le e s, 102 (2009) 084302. 552 13. S. D akopoulou, S. Te zakis, M. Foun oulakis, D. Man za inos, T. Manios, Ul asound-553 induced inac i a ion o g am-nega i e and g am-posi i e bac e ia in seconda y ea ed municipal 554 was ewa e , Ul asonics sonochemis y, 16 (2009) 629-634. 555 14. D. Venie i, E. Ma kogiannaki, E. Cha zisymeon, E. Diamadopoulos, D. Man za inos, 556 Inac i a ion o Bacillus an h acis in wa e by pho oca aly ic, pho oly ic and sonochemical ea men , 557 Pho ochemical & Pho obiological Sciences, (2013). 558 15. R.V. Pe e son, W.G. Pi , The e ec o equency and powe densi y on he ul asonically-559 enhanced killing o bio ilm-seques e ed Esche ichia coli, Colloids and Su aces B: Bioin e aces, 17 560 (2000) 219-227. 561 17  16. A. Moody, G. Ma x, B.G. Swanson, D. Be múdez-Agui e, A comp ehensi e s udy on he 562 inac i a ion o Esche ichia coli unde non he mal echnologies: High hyd os a ic p essu e, pulsed 563 elec ic ields and ul asound, Food Con ol, 37 (2014) 305-314. 564 17. A.-G. Rincón, C. Pulga in, Fe3+and TiO2 sola -ligh -assis ed inac i a ion o E. coli a ield 565 scale: Implica ions in sola disin ec ion a low empe a u e o la ge quan i ies o wa e , Ca alysis 566 oday, 122 (2007) 128-136. 567 18. A. Moncayo-Lasso, J. Sanab ia, C. Pulga in, N. Beni ez, Simul aneous E. coli inac i a ion and 568 NOM deg ada ion in i e wa e ia pho o-Fen on p ocess a na u al pH in sola CPC eac o . A new 569 way o enhancing sola disin ec ion o na u al wa e , Chemosphe e, 77 (2009) 296-300. 570 19. R.J. Bull, F.C. Kop le , Heal h e ec s o disin ec an s and disin ec ion by-p oduc s, AWWA, 571 1991. 572 20. T.M. Olson, P.F. Ba bie , Oxida ion kine ics o na u al o ganic ma e by sonolysis and ozone, 573 Wa e esea ch, 28 (1994) 1383-1391. 574 21. E. O ega-Gómez, M.M.B. Ma ín, B.E. Ga cía, J.A.S. Pé ez, P.F. Ibáñez, Sola Pho o-Fen on 575 o Wa e Disin ec ion: An In es iga ion o he Compe i i e Role o Model O ganic Ma e o 576 Oxida i e Species, Applied Ca alysis B: En i onmen al, (2013). 577 22. R.A. To es, F. Abdelmalek, E. Combe , C. Pe ie , C. Pulga in, A compa a i e s udy o 578 ul asonic ca i a ion and Fen on's eagen o bisphenol A deg ada ion in deionised and na u al wa e s, 579 Jou nal o haza dous ma e ials, 146 (2007) 546-551. 580 23. F. Sciacca, J.A. Rengi o-He e a, J. Wé hé, C. Pulga in, Sola disin ec ion o wild Salmonella 581 sp. in na u al wa e wi h a 18L CPC pho o eac o : De imen al e ec o non-s e ile s o age o ea ed 582 wa e , Sola Ene gy, 85 (2011) 1399-1408. 583 24. J. Rod íguez-Chueca, M.I. Polo-López, R. Mos eo, M.P. O mad, P. Fe nández-Ibáñez, 584 Disin ec ion o eal and simula ed u ban was ewa e e luen s using mild sola pho o-Fen on, Applied 585 Ca alysis B: En i onmen al. 586 25. D. Spuhle , J. And és Rengi o-He e a, C. Pulga in, The e ec o Fe2+, Fe3+, H2O2 and he 587 pho o-Fen on eagen a nea neu al pH on he sola disin ec ion (SODIS) a low empe a u es o 588 wa e con aining Esche ichia coli K12, Applied Ca alysis B: En i onmen al, 96 (2010) 126-141. 589 26. P. Fe nández, J. Blanco, C. Sichel, S. Mala o, Wa e disin ec ion by sola pho oca alysis using 590 compound pa abolic collec o s, Ca alysis Today, 101 (2005) 345-352. 591 27. A.-G. Rincón, C. Pulga in, Absence o E. coli eg ow h a e Fe3+and TiO2 sola 592 pho oassis ed disin ec ion o wa e in CPC sola pho o eac o , Ca alysis oday, 124 (2007) 204-214. 593 28. D.B. Miss ea , J.P. Mu agh, L.W. Gill, The E ec o Da k Pe iods on he UV Pho oly ic and 594 Pho oca aly ic Disin ec ion o Esche ichia coli in a Con inuous Flow Reac o , Jou nal o Sola Ene gy 595 Enginee ing, 135 (2013) 021012-021012. 596 29. S. Giannakis, A.I. Me ino Gamo, E. Da akas, A. Escalas-Cañellas, C. Pulga in, Impac o 597 di e en ligh in e mi ence egimes on bac e ia du ing simula ed sola ea men o seconda y 598 e luen : Implica ions o he inse ed da k pe iods, Sola Ene gy, 98, Pa C (2013) 572-581. 599 30. J. Ndounla, S. Ken ack, J. Wé hé, C. Pulga in, Rele an impac o i adiance ( s. dose) and 600 e olu ion o pH and mine al ni ogen compounds du ing na u al wa e disin ec ion by pho o-Fen on in 601 a sola CPC eac o , Applied Ca alysis B: En i onmen al, 148–149 (2014) 144-153. 602 31. P. Fe nández-Ibáñez, C. Sichel, M. Polo-López, M. de Ca a-Ga cía, J. Tello, Pho oca aly ic 603 disin ec ion o na u al well wa e con amina ed by Fusa ium solani using TiO2 slu y in sola CPC 604 pho o- eac o s, Ca alysis Today, 144 (2009) 62-68. 605 32. OECD Guidelines o Tes ing o Chemicals, Simula ion Tes -Ae obic Sewage T ea men 606 303A, 1999. 607 33. M. Gibbs, A simple me hod o he apid de e mina ion o i on in na u al wa e s, Wa e 608 esea ch, 13 (1979) 295-297. 609 18  34. F. Mendez-A iaga, R.A. To es-Palma, C. Pe ie , S. Esplugas, J. Gimenez, C. Pulga in, 610 Ul asonic ea men o wa e con amina ed wi h ibup o en, Wa e esea ch, 42 (2008) 4243-4248. 611 35. J. Ma ugán, R. an G ieken, C. Pablos, C. So do, Analogies and di e ences be ween 612 pho oca aly ic oxida ion o chemicals and pho oca aly ic inac i a ion o mic oo ganisms, Wa e 613 esea ch, 44 (2010) 789-796. 614 36. P. Bu z, B. Tausche , Eme ging echnologies: chemical aspec s, Food esea ch in e na ional, 615 35 (2002) 279-284. 616 37. P. Fellows, Food p ocessing echnology: P inciples and p ac ice. (2000) 2nd ed., CRC P ess, 617 New Yo k 618 38. E.R. Holm, D.M. S ampe , R.A. B izzola a, L. Ba nes, N. Deame , J.M. Bu kholde , 619 Sonica ion o bac e ia, phy oplank on and zooplank on: Applica ion o ea men o ballas wa e , 620 Ma ine pollu ion bulle in, 56 (2008) 1201-1208. 621 39. T. Leigh on, The acous ic bubble, Access Online ia Else ie , 1994. 622 40. A. Molina-Höppne , W. Dos e , R.F. Vogel, M.G. Gänzle, P o ec i e e ec o suc ose and 623 sodium chlo ide o Lac ococcus lac is du ing suble hal and le hal high-p essu e ea men s, Applied 624 and en i onmen al mic obiology, 70 (2004) 2013-2020. 625 41. C. Pe ie e al, in: T.J. Mason, A. Tiehm, Ad ances in Sonochemis y, Volume 6: Ul asound 626 in En i onmen al P o ec ion, Access Online ia Else ie , 2001 627 42. R.S. Da idson, A. Sa da , J.D. Spence , B. Robinson, Applica ions o ul asound o o ganic 628 chemis y, Ul asonics, 25 (1987) 35-39. 629 43. Y. Ma sumu a, A. Iwasawa, T. Kobayashi, T. Kamachi, T. Ozawa, M. Kohno, De ec ion o 630 High- equency Ul asound-induced Single Oxygen by he ESR Spin- apping Me hod, Chemis y 631 Le e s, 42 (2013) 1291-1293. 632 44. J. Ndounla, D. Spuhle , S. Ken ack, J. Wé hé, C. Pulga in, Inac i a ion by sola pho o-Fen on 633 in pe bo les o wild en e ic bac e ia o na u al well wa e : Absence o e-g ow h a e one week o 634 subsequen s o age, Applied Ca alysis B: En i onmen al, 129 (2013) 309-317. 635 45. F. Za iska, P. D ogui, E.M. El Hachemi, E. Na echoux, E ec o ni a e ions on he 636 e iciency o sonopho ochemical phenol deg ada ion, Ul asonics sonochemis y, 21 (2014) 69-75. 637 46. E. No o, M. Pa ola, Redox mechanisms in hepa ic ch onic wound healing and ib ogenesis, 638 Fib ogenesis & issue epai , 1 (2008) 5. 639 47. M.D. K yszczuk, The e ec o ul asound in a chemo he apeu ic s udy o asci es umo cells, 640 in, Ca holic Uni e si y o Ame ica P ess, Washing on, 1962. 641 48. E. Dahl, Physicochemical aspec s o disin ec ion o wa e by means o ul asound and ozone, 642 Wa e esea ch, 10 (1976) 677-684. 643 49. R.A. To es, G. Sa an akos, E. Combe , C. Pé ie , C. Pulga in, Sequen ial helio-pho o-Fen on 644 and sonica ion p ocesses o he ea men o bisphenol A, Jou nal o Pho ochemis y and 645 Pho obiology A: Chemis y, 199 (2008) 197-203. 646 50. M. Chauhan, E ec o ul asound on he edox eac ions o i on (II) and (III), Indian jou nal o 647 chemis y. Sec . A: Ino ganic, physical, heo e ical & analy ical, 43 (2004) 2098-2101. 648 51. Y.T. Didenko, S. Pugach, Spec a o wa e sonoluminescence, The Jou nal o Physical 649 Chemis y, 98 (1994) 9742-9749. 650 52. C. Pe ie , A. Jeune , J.L. Luche, G. Re e dy, Unexpec ed equency e ec s on he a e o 651 oxida i e p ocesses induced by ul asound, Jou nal o he Ame ican Chemical Socie y, 114 (1992) 652 3148-3150. 653 53. C. Pe ie , M.-F. Lamy, A. F ancony, A. Benahcene, B. Da id, V. Renaudin, N. Gond exon, 654 Sonochemical deg ada ion o phenol in dilu e aqueous solu ions: compa ison o he eac ion a es a 20 655 and 487 kHz, The Jou nal o Physical Chemis y, 98 (1994) 10514-10520. 656 54. C. Pe ie , B. Da id, S. Laguian, Ul asonic deg ada ion a 20 kHz and 500 kHz o a azine 657 and pen achlo ophenol in aqueous solu ion: P elimina y esul s, Chemosphe e, 32 (1996) 1709-1718. 658 19  55. S. Koda, M. Miyamo o, M. Toma, T. Ma suoka, M. Maebayashi, Inac i a ion o Esche ichia 659 coli and S ep ococcus mu ans by ul asound a 500kHz, Ul asonics sonochemis y, 16 (2009) 655-660 659. 661 56. E. Joyce, A. Al‐Hashimi, T. Mason, Assessing he e ec o di e en ul asonic equencies on 662 bac e ial iabili y using low cy ome y, Jou nal o applied mic obiology, 110 (2011) 862-870. 663 57. C.N. Haas, R.S. Engelb ech , Physiological al e a ions o ege a i e mic oo ganisms esul ing 664 om chlo ina ion, Jou nal (Wa e Pollu ion Con ol Fede a ion), (1980) 1976-1989. 665 58. S. B oekman, O. Pohlmann, E.S. Bea dwood, E.C. de Meulenae , Ul asonic ea men o 666 mic obiological con ol o wa e sys ems, Ul asonics sonochemis y, 17 (2010) 1041-1048. 667 59. A. Hulsmans, K. Jo is, N. Lambe , H. Redie s, P. Decle ck, Y. Delaed , F. Olle ie , S. Lie s, 668 E alua ion o p ocess pa ame e s o ul asonic ea men o bac e ial suspensions in a pilo scale wa e 669 disin ec ion sys em, Ul asonics sonochemis y, 17 (2010) 1004-1009. 670 60. A. Al Bsoul, J.P. Magnin, N. Commenges-Be nole, N. Gond exon, J. Willison, C. Pe ie , 671 E ec i eness o ul asound o he des uc ion o Mycobac e ium sp. s ain (6PY1), Ul asonics 672 sonochemis y, 17 (2010) 106-110. 673 61. E. O ega-Gomez, P. Fe nandez-Ibanez, M.M. Balles e os Ma in, M.I. Polo-Lopez, B. 674 Es eban Ga cia, J.A. Sanchez Pe ez, Wa e disin ec ion using pho o-Fen on: E ec o empe a u e on 675 En e ococcus aecalis su i al, Wa e esea ch, 46 (2012) 6154-6162. 676 62. S. Giannakis, E. Da akas, A. Escalas-Cañellas, C. Pulga in, The an agonis ic and syne gis ic 677 e ec s o empe a u e du ing sola disin ec ion o syn he ic seconda y e luen , Jou nal o 678 Pho ochemis y and Pho obiology A: Chemis y, 280 (2014) 14-26. 679 63. A. Rincon, C. Pulga in, Pho oca aly ical inac i a ion o E. coli: e ec o (con inuous–680 in e mi en ) ligh in ensi y and o (suspended– ixed) TiO2 concen a ion, Applied Ca alysis B: 681 En i onmen al, 44 (2003) 263-284. 682 64. V. Raman, A. Abbas, Expe imen al in es iga ions on ul asound media ed pa icle b eakage, 683 Ul asonics sonochemis y, 15 (2008) 55-64. 684 65. E. Joyce, S.S. Phull, J.P. Lo ime , T.J. Mason, The de elopmen and e alua ion o ul asound 685 o he ea men o bac e ial suspensions. A s udy o equency, powe and sonica ion ime on 686 cul u ed Bacillus species, Ul asonics sonochemis y, 10 (2003) 315-318. 687 66. N. Klame h, S. Mala o, A. Agüe a, A. Fe nández-Alba, G. Mailho , T ea men o Municipal 688 Was ewa e T ea men Plan E luen s wi h Modi ied Pho o-Fen on As a Te ia y T ea men o he 689 Deg ada ion o Mic o Pollu an s and Disin ec ion, En i onmen al Science & Technology, 46 (2012) 690 2885-2892. 691 67. J. Rod íguez-Chueca, M.I. Polo-López, R. Mos eo, M.P. O mad, P. Fe nández-Ibáñez, 692 Disin ec ion o eal and simula ed u ban was ewa e e luen s using a mild sola pho o-Fen on, 693 Applied Ca alysis B: En i onmen al, 150–151 (2014) 619-629. 694 695 20  Lis o Tables 696 Table 1 – Hyd aulic cha ac e is ics o p e ious wo ks in CPC eac o s 697 Flow a e (L/min) To al olume (L) Volume- o- low a e a io Illumina ed Volume Volume in he da k Ligh - o- da k a io Fe nandez- Ibañez e al. (2009) 20 14 0.70 32% 68% 0.47 Moncayo- Lasso e al. (2009) 17.5 20 1.14 45% 55% 0.82 Fe nandez- Ibañez e al. (2005) ( a ied low a es) 5, 13, 22.5 11 2.2, 0.85, 0.49 49% 51% 0.96 Rincon & Pulga in (2007) (min, max capaci y) 20.5 37, 70 1.80, 3.41 65%, 34% 35%, 66% 1.86, 0.52 Sciacca e al. (2011) 24.2 18 0.74 83% 17% 4.88 Giannakis e al. (2013) 0.03, 0.06, 0.07 0.7 22.58, 12.28, 9.59 33% 67% 0.49 Ndounla e al. (2013) 2 25 12.5 60% 40% 1.53 698 Table 2 – Pa ame e s in ol ed in he join ea men p ocess 699 Fac o s Values O he pa ame e s Ligh 1000 W/m2Tempe a u e: 30˚C Ul asound 20 W Reci cula ing Flow a e: 4.39 L/h I on 1 ppm T ea ed Volume: 500 mL H2O2 10 ppm Ini ial Popula ion: 106 CFU/mL 700 701 21  Table 3 – Subse s o expe imen s in he s ep-wise cons uc ion o he join h /US/Fe/H2O2 702 ea men p ocess. 703 Expe imen s T ea men cons i uen s 1-2 WW and WW/Fe/H2O2 ‐ Was ewa e wi h no ea men ‐ Was ewa e + Fe/H2O2 3-4 US and US/Fe/H2O2 ‐ Was ewa e +US ‐ Was ewa e +US+Fe/H2O2 5-6 h and h /Fe/H2O2 ‐ Ligh ‐ Ligh +/Fe/H2O2 (pho o-Fen on) 7-8 h /US and h /US/Fe/H2O2 ‐ Ligh +US ‐ US+pho o-Fen on 704 Table 4 – O e iew o he in es iga ion o he ope a ional pa ame e s 705 Fac o s1 Le el 1 Le el 2 Le el 3 Hyd aulic Pump pm 33 66 99 No. o Illumina ed essels 1 2 3 Was ewa e olume (mL) 500 600 700 En i onmen al Tempe a u e (˚C) 10 20 30 Ligh In ensi y (W/m2) 800 1000 1200 Fen on / Ul asound H2O2 Concen a ion (ppm) 5 10 20 Fe Concen a ion (ppm) 0.5 1 2 US Acous ic Powe (W) 10 20 40 1Cen al alues a e anno a ed wi h bold. 706 707 22  708 Table 5 – Hyd aulic calcula ions on he eac o se -up 709 710 Inc easing eci cula ion speed om 33 o 99 pm (1.87 o 4.39 L/h) Reac o s333 pmReac o s366 pmReac o s399 pm 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 Ligh 230mL7.38min46%Ligh 230mL4.01min46%Ligh 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% To al500mL16.04min100%To al500mL8.72min100%To al500mL6.83min100% Inc easing illumina ed olume om 1 eac o o 3 (75 o 230 mL) Reac o s199 pmReac o s299 pmReac o s399 pm 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 Ligh 75mL1.03min15%Ligh 150mL2.05min30%Ligh 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% To al500mL6.83min100%To al500mL6.83min100%To al500mLmL6.83min100% Inc easing o al ea ed olume om 500 o 700 mL Reac o s399 pmReac o s399 pmReac o s399 pm 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 Ligh 230mL3.14min46%Ligh 230mL3.14min38%Ligh 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% To al500mL6.83min100%To al600mL8.20min100%To al700mL9.57min100% 24  Lis o Figu es 712 713 Figu e 1 – Expe imen al se -up, consis ing o he illumina ed a ea (Sun es appa a us), he eci cula ion pump, 714 he (da k) sonica ion essel and he empe a u e con ol ( he mos a ). The low di ec ion is clockwise, wa e is 715 in oduced a su ace le el and sampled om he bo om o he essel. 716 717 718 Figu e 2 – Expe imen al esul s om he coupling o pho o-Fen on and sonica ion. a) Expe imen s 1-2 (WW and 719 WW/Fe/H2O2), b) Expe imen s 3-4 (US and US/Fe/H2O2), c) Expe imen s 5-6 (h and h /Fe/H2O2) and d) 720 Expe imen s 7-8 (h /US and h /US/Fe/H2O2). e) Long- e m inac i a ion e en s o 48 h ( ime axis ini ia es in he 721 4-h ma k, a e ea men ). 722 723 25  724 Figu e 3 – Sugges ion o he added ac ions sonica ion has owa ds bac e ial inac i a ion, when coupled wi h 725 pho o-Fen on. The known pho o-Fen on mechanisms sugges ed by li e a u e a e no displayed. 726 727