Enhanced nitrogen removal of low carbon wastewater in denitrification bioreactors by utilizing industrial waste toward circular economy

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY-NC-ND 4.0 h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by u ilizing indus ial was e owa d ci cula economy © Else ie L d. Accep ed e sion (Final d a ) Kiani, Sepideh; Kujala, Ka ha ina; Pulkkinen, Jani; Aal o, Sanni L.; Suu näkki, Su i; Kiu u, Tapio; Tii ola, Ma ja; Klø e, Bjø n; Ronkanen, Anna-Kaisa Kiani, S., Kujala, K., Pulkkinen, J., Aal o, S. L., Suu näkki, S., Kiu u, T., Tii ola, M., Klø e, B., & Ronkanen, A.-K. (2020). Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by u ilizing indus ial was e owa d ci cula economy. Jou nal o Cleane P oduc ion, 254, A icle 119973. h ps://doi.o g/10.1016/j.jclep o.2020.119973 2020 Jou nal P e-p oo Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by u ilizing indus ial was e owa d ci cula economy Sepideh Kiani, Ka ha ina Kujala, Jani Pulkkinen, Sanni L. Aal o, Su i Suu näkki, Tapio Kiu u, Ma ja Tii ola, Bjø n Klø e, Anna-Kaisa Ronkanen PII: S0959-6526(20)30020-2 DOI: h ps://doi.o g/10.1016/j.jclep o.2020.119973 Re e ence: JCLP 119973 To appea in: Jou nal o Cleane P oduc ion Recei ed Da e: 30 Augus 2019 Re ised Da e: 12 Decembe 2019 Accep ed Da e: 2 Janua y 2020 Please ci e his a icle as: Kiani S, Kujala K, Pulkkinen J, Aal o SL, Suu näkki S, Kiu u T, Tii ola M, Klø e Bjø, Ronkanen A-K, Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by u ilizing indus ial was e owa d ci cula economy, Jou nal o Cleane P oduc ion (2020), doi: h ps:// doi.o g/10.1016/j.jclep o.2020.119973. This is a PDF ile o an a icle ha has unde gone enhancemen s a e accep ance, such as he addi ion o a co e page and me ada a, and o ma ing o eadabili y, bu i is no ye he de ini i e e sion o eco d. This e sion will unde go addi ional copyedi ing, ypese ing and e iew be o e i is published in i s inal o m, bu we a e p o iding his e sion o gi e ea ly isibili y o he a icle. Please no e ha , du ing he p oduc ion p ocess, e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. © 2020 Published by Else ie L d. Sepideh kiani: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion Anna-Kaisa Ronkanen: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion Bjö n Klö e: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion Ka ha ina Kujala: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - O iginal D a , W i ing - Re iew & Edi ing, Visualiza ion, Supe ision Jani Pulkkinen: Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew & Edi ing, Tapio Kiu u: In es iga ion, Resou ces, P ojec adminis a ion Sanni L. Aal o: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew & Edi ing, Visualiza ion Su i Suu näkki: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew & Edi ing, Visualiza ion Ma ja Tii ola: Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces, W i ing - Re iew & Edi ing, Visualiza ion, Supe ision, P ojec adminis a ion 1 Enhanced ni ogen emo al o low ca bon was ewa e in deni i ica ion bio eac o s by u ilizing indus ial was e owa d ci cula economy Sepideh Kiani a *, Ka ha ina Kujala a , Jani Pulkkinen b , Sanni L. Aal o c,d , Su i Suu näkki c , Tapio Kiu u b , Ma ja Tii ola c , Bjø n Klø e a and Anna-Kaisa Ronkanen a a Wa e , Ene gy and En i onmen al Enginee ing Resea ch Uni , Facul y o Technology, P.O. Box 4300, FI- 90014 Uni e si y o Oulu, Finland b Na u al Resou ces Ins i u e Finland, Su on ie 9A, 40500 Jy äskylä, Finland c Depa men o Biological and En i onmen al Science, Nanoscience Cen e , 40014 Uni e si y o Jy äskylä, Finland d Depa men o En i onmen al and Biological Sciences, Uni e si y o Eas e n Finland, P.O. Box 1627, 70211 Kuopio, Finland *Co esponding au ho : Sepideh Kiani (Email: sepide[email p o ec ed]) 1 Abs ac 1 Aquacul u e needs p ac ical solu ions o nu ien emo al o achie e sus ainable ish p oduc ion. Passi e 2 deni i ying bio eac o s may p o ide an ecological, low-cos and low-main enance app oach o was ewa e 3 ni ogen emo al. Howe e , inno a i e o ganic ma e ials a e needed o enhance ni a e emo al om he low 4 ca bon e luen s in in ensi e eci cula ing aquacul u e sys ems (RAS). In his s udy, we es ed h ee 5 addi ional ca bon sou ces, including biocha , d ied Sphagnum sp. moss and indus ial po a o esidues, o 6 enhance he pe o mance o woodchip bio eac o s ea ing he low ca bon RAS was ewa e . We assessed 7 ni a e (NO 3- ) emo al and mic obial communi y composi ion du ing a one-yea in si u column es wi h eal 8 aquacul u e was ewa e . We ound no signi ican di e ences in he NO 3- emo al a es be ween he 9 woodchip-only bio eac o and bio eac o s wi h a zone o biocha o Sphagnum sp. moss (maximum emo al 10 a e 31-33 g NO 3- -N m -3 d -1 ), bu po a o esidues inc eased NO 3- emo al a e o 38 g NO 3- -N m -3 d -1 , wi h 11 s able annual educ ion e iciency o 93%. The eadily a ailable ca bon eleased om po a o esidues 12 inc eased NO 3- -N emo al capaci y o he bio eac o e en a highe in low concen a ions (>52 mg L -1 ). The 13 mic obial communi y and i s p edic ed unc ional po en ial in he po a o esidue bio eac o di e ed ma kedly 14 om hose o he o he bio eac o s. Adding po a o esidues o woodchip ma e ial enabled smalle bio eac o 15 size o be used o NO 3- emo al. This s udy in oduced indus ial po a o by-p oduc as an al e na i e ca bon 16 sou ce o he woodchip deni i ica ion p ocess, and he encou aging esul s may pa e he way owa d 17 g ow h o blue bioeconomy using he RAS. 18 19 Keywo ds: Reci cula ing aquacul u e sys em, woodchip bio eac o , ca bon sou ce, po a o esidues, ni a e, 20 mic obial communi y 21 22 23 24 25 26 27 2 1 In oduc ion 28 Reci cula ing aquacul u e sys ems (RAS) a e en i onmen ally iendly solu ions ha aim o achie e ze o 29 was e om ish p oduc ion. Al hough RAS ha e been used o mo e han 10 yea s in di e en coun ies, 30 including wo la ges RAS in Finland wi h a p oduc ion capaci y o o e 4000 ons, ni a e (NO 3- ) emo al is 31 s ill a c i ical challenge (Pulkkinen e al., 2018). Remo al o NO 3- is a challenge as aquacul u e was ewa e 32 has low ca bon (C) bu high ni ogen (N) concen a ions. A ew p e ious s udies ha e examined he use o 33 deni i ying bio eac o s o ea ing aquacul u e e luen . So a , such s udies ha e ocused on RAS e luen s 34 wi h high chemical oxygen demand (COD) (Lepine e al., 2016), added bica bona e (HCO -3 ) o inle wa e 35 ( on Ahnen e al., 2016b) and dilu ed e luen om an ou doo ish a m wi h low eci cula ion in ensi y and 36 low NO 3- -N concen a ion (~6 mg L -1 ) ( on Ahnen e al., 2018, 2016a). In con as , ea men o highly 37 in ensi e indoo RAS e luen s wi h low COD (12.9 ± 1.8 mg L -1 ) and high NO 3- -N concen a ion (>50 mg 38 L -1 ) has ecei ed li le a en ion. 39 In deni i ying bio eac o s, ni ogen (N) is emo ed by he e o ophic deni i ie s con e ing NO 3- o ni ogen 40 gas unde anoxic condi ions. Unde ni a e- ich condi ions, his p ocess depends on he a ailabili y o he 41 ca bon sou ce as he o ganic elec on dono (Wang and Chu, 2016). Ex e nal ca bon sou ces, such as ace a e 42 o me hanol, a e o en supplied o he sys em o achie e e icien deni i ica ion (Che chi e al., 2009). 43 Howe e , he cos o ca bon addi ion is ypically high (Zhang e al., 2016) and he p ocess needs egula ion 44 o p e en o e - o unde -dosing o he liquid ca bon sou ces (Roche e al., 2015). Solid ca bon sou ces can 45 p o ide a cos -e ec i e al e na i e o he classical ca bon sou ces men ioned abo e. In ecen yea s, esea ch 46 has ocused on solid ca bon sou ces wi h high quali y, op imal e iciency and slow- elease abili y in he 47 ea men o excessi ely ni a e-con amina ed wa e , pa icula ly su ace wa e (Beu el e al., 2016) and 48 g oundwa e (Zhang e al., 2012). Wood-pa icle p oduc s (e.g. woodchip and sawdus ) ha e been widely 49 used, due o hei abili y o supply ca bon o he deni i ica ion p ocess o 5-15 yea s and hus allow good 50 NO 3- emo al wi h minimum bio eac o main enance (Schippe e al., 2010). Howe e , he la ge space 51 equi emen o ull-scale woodchip bio eac o s has p omp ed e o s o enhance he deni i ica ion a e by 52 using inno a i e na u al ca bon sou ces (Tangsi e al., 2017). Inexpensi e indus ial ood by-p oduc s, such 53 3 as indus ial po a o esidue, could ha e high po en ial o be u ilized in iden i ying bio eac o o enhance 54 ni a e emo al. Po a o indus ies can gene a e 20-25 % was e om peeling, imming and cu ing p ocesses 55 (Liang and McDonald, 2014). 56 This s udy examined he use o a deni i ying bio eac o o ea indoo in ensi e RAS e luen wi h low 57 COD and high NO 3- concen a ion, as pa o he unique RAS esea ch pla o m (see Pulkkinen e al., 2018), 58 and compa ed di e en ca bon sou ces, including po a o esidue, o imp o ing he ni ogen emo al 59 pe o mance o woodchip bio eac o s. The o e all aim was o e alua e he pe o mance o deni i ying 60 bio eac o s in emo ing NO 3- om aquacul u e was ewa e wi h low COD o a pe iod o o e one yea . 61 Speci ic objec i es we e o (1) s udy he sui abili y o wood-based bio eac o s o ea ing RAS e luen , (2) 62 assess whe he he NO 3- emo al pe o mance o woodchip p ocess can be enhanced by addi ional ca bon 63 sou ces, (3) o assess he e ec o di e en ca bon sou ces on he mic obial communi y composi ion in 64 di e en compa men s o he bio eac o s, and (4) o iden i y dominan bac e ia and hei unc ional po en ial 65 in he bio eac o s s udied. The in en ion was o ind solu ions o imp o ing wa e ea men and o 66 enhancing NO 3- emo al in he eci cula ing aquacul u e sys ems. 67 2 Ma e ial and me hods 68 2.1 RAS e luen wa e quali y 69 The s udy was conduc ed a he Laukaa ish a m o he Na u al Resou ces Ins i u e Finland (LUKE) in 70 cen al Finland, in he esea ch pla o m examining RAS. The RAS design is desc ibed in de ail in Pulkkinen 71 e al. (2018). In b ie , e luen was ob ained om a RAS consis ing o a eed collec o uni , swi l sepa a o , 72 d um il e (60 µm mesh) and ixed bed bio eac o , ollowed by a mo ing bed bio eac o and a ickling 73 il e . In o de o p e en any changes in wa e chemis y, mic obiology o wa e empe a u e, all es s we e 74 pe o med using he na u al RAS e luen . The e luen is cha ac e ised by low ca bon (15.3 mg L -1 on 75 a e age), bu high N con en (mean NO 3- -N con en 34.7 mg L -1 ) (Table 1). Due o he e icien ni i ica ion 76 uni be o e he bio eac o s, NO 3- is domina ing N ac ion. 77 Table 1. Mean in low wa e quali y pa ame e s (SD = s anda d de ia ion, n = numbe o sample) 78 4 Wa e quali y pa ame e s In low (mean ± SD) n To al o ganic ca bon (mg L - 1 ) 15.3 ± 2.1 5 Dissol ed o ganic ca bon (mg L - 1 ) 14 ± 1.3 5 Chemical oxygen demand (mg L - 1 ) 12.9 ± 1.8 5 Biological oxygen demand (mg L - 1 ) 3.8 ± 2.2 13 Ni a e-ni ogen (mg L - 1 ) 34.7 ± 15.6 27 Ni i e-ni ogen (mg L - 1 ) 0.1 ± 0.06 30 Ammonium-ni ogen (mg L - 1 ) 0.5 ± 0.2 30 Dissol ed oxygen (mg L - 1 ) 8.1 ± 1.7 29 pH 6.9 ± 0.2 28 Oxida ion - educ ion po en ial ( Eh , mV) 178.6 ± 60.4 35 Alkalini y (mg CaCO 3 L - 1 ) 54.2 ± 18 25 Sulpha e (mg L - 1 ) 10.5 ± 3.2 24 2.2 Bio eac o design 79 The pe o mance o deni i ying bio eac o s was s udied in ou anspa en ac ylic columns (0.1 m diame e 80 × 0.32 m high) wi h upwa d low di ec ion applying a heo e ical e en ion ime (HRT) o 48 h a con olled 81 empe a u e (15.5±0.8°C) (Fig. 1). In each column, he eac i e media we e placed on op o an ine qua z 82 g a el bed, om which hey we e sepa a ed by plas ic ne ing wi h 2 mm po e size, o p e en clogging wi h 83 ma e ials con aining o ganic ma e . A cons an in low a e o 0.6 mL min -1 was applied o each bio eac o 84 o 346 days, using a pe is al ic pump. The upwa d low di ec ion and he qua z g a el laye a he base o 85 he columns p e en ed he de elopmen o p e e en ial low pa hways and ensu ed uni o m dis ibu ion o 86 low in o he columns. The columns consis ed o packed-media zones (zone 1, zone 2, zone 3) con aining 87 woodchips, indus ial po a o was e, biocha o d ied Sphagnum sp. moss in he a ios shown in Fig. 1. The 88 packed-media has no been eplaced du ing he s udy pe iod. All bio eac o s wi h addi ional laye con ain 89 same o al olume o woodchips. Howe e , Sphagnum sp. moss was mixed wi h woodchips in he zone 2, 90 due o i s di e en cha ac e is ic and small pa icle size dis ibu ion. I is well known ha na u al pea has 91 5 ypically low hyd aulic conduc i i y (e.g. Ronkanen and Klø e 2005), which could cause isks in longe 92 HRT o e en clogging o he bio eac o . In o de o a oid his, moss was mixed wi h woodchips. The 93 packed-media zones we e sepa a ed om he ou le ee wa e zone by a ixed pe o a ed PVC pla e 94 ( hickness 5 mm) a a heigh o 4.5 cm om he op o he column. The columns we e sealed a bo h ends o 95 p o ide con olled condi ions. 96 The selec ed ca bon sou ces had di e en C/N a ios, anging om 28 o 249 (Table 2). Woodchips had he 97 highes C/N a io, bu biocha con ained he highes amoun o ca bon. The used woodchips we e ob ained 98 locally om esh bi ch ees (p o ided by he ene gy company Vapo G oup). The a e age woodchip size 99 was a ound 3 cm × 1.5 cm × 0.4 cm and mean po osi y 63%. The Sphagnum sp. moss used was common 100 mi e lo a p o ided by Vapo G oup. The biocha (po osi y 46%) was ob ained om RPK Hiili Oy. The 101 po a o ma e ial es ed comp ised indus ial esidues om POHJOLAN PERUNA Oy wi h a d y ma e 102 con en o 12% (de e mined a e d ying he ma e ial a 105°C o 24 h). 103 P io o he expe imen s, solid ma e ials (woodchips and biocha ) we e washed wi h dis illed wa e and 104 sa u a ed o 48 h. In o de o p e en e men a ion, he po a o esidues we e kep in he eeze p io o use. 105 The ozen po a o esidues we e de os ed a oom empe a u e o 8 h be o e he es . 106 12 s a , bu only a e s able deni i ica ion a es a e es ablished and low ni i e concen a ions a e de ec ed in 211 he ou low. 212 The in low NH 4+ -N concen a ion anged be ween 0.17-1.0 mg L -1 (Table 1; Fig. S1b). Low NH 4+ -N 213 p oduc ion was de ec ed in all bio eac o s, wi h ou low concen a ions o 0.8±0.5 mg L -1 , 0.9±0.5 mg L -1 , 214 0.9±0.6 mg L -1 and 3.8±3.4 mg L -1 in BR1, BR2, BR3 and BR4, espec i ely. Less han 2 mg L -1 o NH 4+ -N 215 was eco ded in he i s h ee weeks in BR1-BR3 (Fig. S1b). Howe e , he bio eac o wi h po a o esidues 216 (BR4) showed ela i ely high NH 4+ -N, wi h a mean concen a ion o 10 mg L -1 , in he i s 10 days o he 217 expe imen , bu i hen declined o lowe han 4 mg L -1 o each he backg ound le el. The con inuous 218 p oduc ion o ammonium in BR4 indica es he occu ence o dissimila i y ni a e educ ion o ammonium 219 (DNRA). In gene al, a educing en i onmen and high TOC/NO 3- a io (1400/15-110/16 in BR4; days 1-70) 220 can indica e he occu ence o DNRA (K a e al., 2014; an Rijn e al., 2006). DNRA has also been 221 obse ed in p e ious woodchip bio eac o s udies (Lu e al., 2013; Zhao e al., 2018). Reducing condi ions, 222 indica ed by Eh alues, we e also seen in his s udy, which led he sys em o SO 42- educ ion (Fig. 4). 223 In he s a -up phase, all bio eac o s eleased DOC. The a e o elease was highes in BR4, wi h ou low 224 concen a ions o 1380 mg L -1 measu ed on day 6 a e s a -up (Table. S1). The DOC elease om he o he 225 bio eac o s was much lowe (<100 mg L -1 ; Table S1). Wi hin 70 days a e s a -up, ou low DOC 226 concen a ion dec eased o 81 mg L -1 in BR4 and o he backg ound le el (14 ± 1.3 mg DOC L -1 ) in BR1-227 BR3 (Table S1). Ini ial ca bon con en lush-ou is common in bio eac o s. The s a -up COD concen a ion 228 in he ou low anged 59-940 mg L -1 in BR1-BR4 (Table. S1) exceeding empo a ily he maximum 229 concen a ion o 42 mg L -1 obse ed in Finnish i e s (Niemi and Raa eland, 2007). Howe e , s a -up phase 230 o he woodchip bio eac o is sho compa ed o es ima ed li e ime (5-15 yea s), so he po en ial pollu ion o 231 ca bon is mino compa ed o he amoun o ni ogen emo ed. Lepine e al. (2016) epo ed an 232 app oxima ely 50-day lush-ou pe iod o a plywood bio eac o ea ing aquacul u e e luen a HRT o 42 h. 233 Somewha highe ca bon leaching (200 mgL -1 ) has been epo ed o bio eac o s packed wi h esh 234 woodchips and a mix u e o woodchips and biocha (Hassanpou e al., 2017; Hoo e e al., 2016). Release 235 o high DOC concen a ions o ecipien wa e bodies om use o bio eac o s as an end-o -pipe ea men 236 can ad e sely a ec aqua ic ecosys ems, e.g. by causing a DO concen a ion educ ion, ligh and empe a u e 237 13 changes (P ai ie, 2008; Solomon e al., 2015), esul ing in lowe ish p oduc ion (S asko e al., 2012). Hence, 238 a si es go e ned by s ic egula ions o when ecycling ou low o ish a ms, high DOC migh need o be 239 con olled. Schippe e al. (2010) iden i ied HRT as a ac o con olling he ini ial magni ude o DOC 240 deple ion and i s du a ion in wood-based bio eac o s. Howe e , he ac ha ca bon was mo e eadily 241 eleased om po a o esidues han om he o he ca bon sou ces used in his s udy p o es ha HRT is no 242 he only con olling ac o and ha ca bon quali y also plays a key ole. In he p esen s udy, he e was 243 signi ican ly lowe ou low DOC concen a ion o 53, 68 and 81 mg L -1 in bio eac o s BR1, BR2 and BR3, 244 which can be pa ly explained by highe ni a e loading (Hassanpou e al., 2017) and pa ly by he ype o 245 ca bon sou ce used. Dependence o TOC leaching and a ia ions in NO 3- -N concen a ion ha e also been 246 epo ed by Zhao e al. (2018). In o de o con ol he ca bon con en due o leaching, i is ecommended o 247 conside pos -bio eac o s ea men uni s (e.g. cons uc ed we land, sand il e ) o eci cula ing he s a -up 248 e luen back o he bio eac o (Schippe e al., 2010). 249 The SO 42- concen a ions we e on a e age highe in he ou low han in he in low wa e s o BR1 and BR2, 250 indica ing leaching o p oduc ion o SO 42- (Fig. 4). This esul ed in cumula i e leaching/p oduc ion o 165 g 251 and 474 g SO 42- in BR1 and BR2, espec i ely, o he whole s udy pe iod. In con as , SO 42- we e on a e age 252 lowe in ou low han in in low wa e s o BR3 and BR4 (Fig. 4), indica ing SO 42- educ ion/ emo al. 253 Cumula i e SO 42- emo al o 350 g and 546 g was obse ed in BR3 and BR4, espec i ely, o he whole 254 s udy pe iod. SO 42- leaching/ emo al inc eased he SO 42- concen a ion in he ou low by up o 20% 255 compa ed wi h he cumula i e in low SO 42- o 2.6 kg. Sulpha e leaching/p oduc ion indica ed he po en ial o 256 in e nal sulphu cycling in bio eac o s wi h incomple e N emo al. BR1 and BR2 had incomple e ni a e 257 emo al du ing he s udy pe iod due o sulphide e-oxida ion o sulpha e by sulphu oxidizing bac e ia 258 (SOB), which can use oxygen o ni a e as elec on accep o (Faulwe e e al., 2009) (Fig. S1 and Fig. 3). 259 Sulpha e p oduc ion was obse ed p e iously by Lepine e el. (2016) o a woodchip bio eac o wi h 260 incomple e N emo al. Howe e , highe ni a e emo al in BR3 and BR4 combined wi h hei educed 261 condi ions (Fig.4) a o ed sulpha e educ ion. 262 14 Fig. 4. Sulpha e educ ion/ emo al (+ alues) and leaching/p oduc ion (- alues) in bio eac o s BR1-BR4 263 o e ime a di e en edox po en ial alues (Eh) in in low and ou low o each bio eac o . 264 Redox po en ial was on a e age +340, +354, +312 and +181 mV in BR1, BR2, BR3 and BR4, espec i ely 265 (Fig. 4), indica ing mo e oxidising condi ions in BR1-BR3 and mo e educing condi ions in BR 4. I is well-266 known ha deni i ica ion and mic obial sulpha e emo al cause decline in edox po en ial and ise in pH 267 (Jog and Pa y., 2006). In BR4, o he en i e s udy pe iod when ou le Eh educed om 412 o 116 mV, he 268 pH ended o inc ease abou 2.2 pH uni s ( om 4.6-6.82) (Fig. S 5). Simila ly, in BR1-3 by dec easing he 269 ou le edox po en ial, he pH inc eased 0.89,1.65 and 1.4 pH uni s, espec i ely . 270 In low wa e pH was a he s able h oughou he expe imen (6.5-7.5) (Fig. 5). Ou low pH o bio eac o s 271 du ing s a -up was 6, 4.3, 5.2 and 3.8 in BR1 BR2, BR3 and BR4, espec i ely. I was hus lowe han 272 in low pH in he ea ly s ages o he expe imen , mos likely as a esul o elease o o ganic acids om he 273 packed ma e ials (Fig. 5). All bio eac o s showed lowe alkalini y in ou low han in in low du ing he s a -274 up pe iod (Fig. 5). A e 2-5 weeks, alkalini y p oduc ion was obse ed in all bio eac o s. 275 15 3.2 Fac o s a ec ing ni a e emo al in woodchip bio eac o s 276 The esul s o one-way ANOVA showed ha NO 3- emo al a es o whole s udy pe iod did no di e 277 signi ican ly be ween BR1, BR2 and BR 3 (p=0.75), while ni a e emo al in BR4 was highe (Fig. 2d-2h). 278 In he i s h ee mon hs o he expe imen , when in low NO 3- -N concen a ion a ied be ween 15 and 52 mg 279 L -1 , all bio eac o s showed simila emo al a es (Fig. 2). A e ha , he bio eac o s esponded di e en ly o 280 inc easing NO 3- -N in low concen a ions, e.g. he emo al a e declined in BR1-BR3 bu inc eased in BR4 281 (Fig. 2). BR4 eached i s maximum emo al a e o 38 g NO 3- -N m -3 d -1 a he highes NO 3- -N in low 282 concen a ion (70 mg L -1 ; days 152-184), whe eas BR1, BR2 and BR3 had a emo al a e o 9, 13 and 12 g 283 NO 3- -N m -3 d -1 , espec i ely (Fig. 2). Those di e ences pe sis ed un il day 250, a e which all eac o s again 284 had simila s able emo al a es o a ound 15 g NO 3- -N m -3 d -1 un il he end o he expe imen . Simila ly o 285 emo al a e, he NO 3- emo al e iciency in BR1-BR3 showed luc ua ions h oughou he s udy pe iod (Fig. 286 2e and 2g). Howe e , BR4 eached s able emo al e iciency o 93% a e a pe iod o luc ua ion a s a -up 287 (Fig. 2h). 288 The wide ange o NO 3- emo al a es (3-38 g NO 3- -N m -3 d -1 ) eco ded in all bio eac o s ollowed he NO 3- -289 N in low concen a ion luc ua ions. High emo al a e in all bio eac o s occu ed when he in low had high 290 Fig. 5 . Alkalini y p oduc ion (+ alues) and in low and ou low pH in bio eac o s (BR1-BR4). 16 NO 3- -N concen a ions. This is consis en wi h p e ious indings ha in low concen a ions con ol emo al 291 a e (e.g. Schippe e al., 2010; Addy e al., 2016). 292 In he p esen s udy, NO 3- emo al a e in BR4 inc eased signi ican ly wi h inc easing NO 3- -N in low 293 concen a ion du ing he en i e s udy pe iod (R 2 = 0.93; emo al a e = 0.6 × in luen ni a e concen a ion - 294 1.85) (Fig. 6). This eg ession illus a ed he ac ual ela ionship be ween in low NO 3- -N concen a ion and 295 emo al a e by excluding NO 3- -N limi ed e en s (NO 3- -N concen a ion <0.5 mg L -1 ) (Addy e al., 2016). 296 Likewise, bio eac o s BR1-BR3 showed a simila esponse o NO 3- -N when days 152-212, wi h high NO 3- -N 297 concen a ion (55-70 mg L -1 ), we e excluded om he da a (Fig. 6). The sha ply decline in NO 3- -N emo al 298 du ing days 152-212 was caused due o exceeding he maximum deni i ica ion capaci y in hose bio eac o s. 299 This indica es ha NO 3- emo al in BR1-BR3 was con olled by an independen pa ame e a high NO 3- -N 300 concen a ions. The elease a e o deg adable ca bon om he packed media p esumably con olled NO 3- 301 emo al in his concen a ion ange (>52 mg L -1 ) (Schippe e al., 2010). Hence, he ype o ca bon sou ce 302 used in deni i ying bio eac o s can con ol NO 3- emo al, by p o iding mo e ca bon a ailabili y and 303 di e en mic obial composi ion (Xu e al., 2018; Tangsi e al., 2017). Obse ed DOC in he bio eac o s 304 showed ha ca bon was much mo e eadily eleased om po a o esidues han om any o he o he ca bon 305 sou ces es ed (Table S1). The easily soluble ca bon in po a o esidues esul ed in apid o ma ion o a 306 complex mic obial communi y s uc u e wi h s ong adap i e g ow h o he new en i onmen (Zhao e al., 307 2018). 308 309 17 The maximum NO 3- emo al a es obse ed in his s udy we e g ea e han hose p e iously epo ed (22 g 310 NO 3- -N m -3 d -1 ) (Da id e al., 2015; Schippe e al., 2010). This could be due o a combina ion o op imal 311 ac o s: su icien HRT (Lepine e al., 2016; Tangsi e al., 2017) as a esul o dis ibu ed upwa d low 312 (sec ion 2.2) combined wi h high NO 3- in low concen a ion (Schippe e al., 2010), he o ganic C 313 compounds used (Gibe e al., 2008) and wa e empe a u e (Addy e al., 2016), he e 15.5 ± 1 °C (mean ± 314 SD). A emo al a e o >39 g NO 3- m -3 d -1 epo ed by Lepine e al. (2016) o compa able wa e quali y was 315 associa ed wi h high COD:NO 3- a io (0.86-1.66) in ea ed was ewa e . This a io can p o ide 42% COD 316 equi ed o deni i ica ion. The COD:NO 3- a io has been epo ed o be a signi ican pa ame e a ec ing 317 deni i ica ion in bio eac o s (Ja a i e al., 2015). Howe e , in he p esen s udy in low COD p o ided less 318 han 8% o he C/N equi ed o comple e NO 3- educ ion (Na kis e al., 1979). Hence, he epo ed NO 3- 319 emo al a es in his s udy ep esen he ne alues wi hou a con ibu ion om in low COD. Enhancing 320 ni a e emo al e iciency wi h di e en ca bon subs a es has been in es iga ed p e iously (Gebe e al., 321 2008; Schippe e al., 2010; Hashemi e al., 2011). Hashemi e al., (2011) imp o ed ni a e emo al o 36% 322 in wood bio eac o o 65%, 56 % and 77 % by u ilizing ba ley s aw, ice husk and da e palm lea , 323 espec i ely. Gebe e al., (2008) epo ed so wood (b anches and ba k wi h small amoun s o lea es om a 324 a ie y o ees) as op pe o ming subs a e in deni i ica ion e iciency (>98%) wi h deni i ica ion a e o ~ 325 Fig. 6 . Ni a e emo al a e e sus ni a e in luen loading in BR1- 4 o he s udy pe iod o 346 days. 18 17 g NO 3- -N m -3 d -1 . Howe e , o he in es iga ed ma e ials such as mix u e o wood chips, sh edded ba k and 326 opsoil, compos (ob ained om he biological decomposi ion o o ganic was es – wood immings, lea es, 327 o en ege ables and ood sc aps) and willow woodchips iden i ied as unsui able ca bon sou ces (see Gebe 328 e al., 2008). Wa neke e al., (2011) epo ed ni a e emo al o ~ 6.5, 6.2 and 3.5 g NO 3- -N m -3 d -1 o whea 329 s aw, maize and g een was e ma e ials, espec i ely compa e o he emo al a e o 1.3 g NO 3- -N m -3 d -1 in 330 so wood (pine) bio eac o o 2- old lowe ni a e inle concen a ion han used in his s udy. Howe e , 331 addi ional po a o esidue o woodchip bio eac o inc eased 13% o ni a e emo al o 38 g NO 3- -N m -3 d -1 332 which is ema kably highe han epo ed emo al abo e. 333 3.3 Mic obial communi y composi ion and p ocess po en ial in he bio eac o s 334 A o al o 9261 quali y- il e ed sequences pe lib a y we e ob ained om wa e and solid samples om he 335 ou bio eac o s (Table 3). Lib a y co e age was ≥94% in all cases, indica ing ha he sequencing dep h was 336 su icien . The numbe o obse ed and Chao 1-es ima ed OTUs was signi ican ly lowe (p<0.001) in il e ed 337 wa e and solid ma e ial om BR4 han in co esponding samples om BR1-BR3. The Shannon di e si y 338 index was also signi ican ly lowe (p<0.001) in BR4 (4.5) han in BR1-BR3. 339 The mic obial communi y in BR4 di e ed s ongly om he mic obial communi y in BR1-BR3 (Figs. S 2A). 340 Smalle di e ences we e de ec ed be ween he mic obial communi ies in BR1-BR3 and be ween wa e and 341 solid samples om all bio eac o s (Figs. S2 B and C). In solid ma e ial, di e ences we e obse ed be ween 342 mic obial communi ies in zone 3 (i.e. op-laye woodchip) and in zone 2 in BR1, BR2 and BR4 (con aining 343 biocha , Sphagnum sp. moss and po a o esidues, espec i ely) bu no BR3 (con aining woodchips) (Fig. 1). 344 In wa e , he di e ences we e much less p onounced (Figs. S2 B and C). 345 Table 3. P oka yo ic di e si y in bio eac o s BR1-BR4. Numbe s o sequences a e aken om he o iginal 346 OTU ables, while all o he di e si y indica o s a e based on OTU ables a i ied a a dep h o 4098 347 sequences. A e age alues o 1-2 eplica es pe sampling poin a e shown. Zone 2 and zone 3 e e o he 348 ca bon sou ce ma e ial es ed and he op-laye woodchip, espec i ely, as indica ed in Fig. 1 349 No. o No. o Co e age OTUs OTUs Shannon 19 sequences samples (%) ichness (obse ed) ichness (es ima ed) a BR 1: Woodchip/ Biocha Wa e Zone 2 8 550 2 95 441 802 4.64 Zone 3 7 310 2 95 468 761 4.68 Solid Zone 2 6 844 2 94 496 827 4.77 Zone 3 4 935 2 95 398 739 4.36 BR 2: Woodchip/ Sphagnum Wa e Zone 2 0 Zone 3 7 500 1 95 450 821 4.67 Solid Zone 2 7 358 1 96 383 697 4.42 Zone 3 6 711 2 96 354 674 4.2 BR 3: Woodchip/ woodchip Wa e Zone 2 8 198 2 95 433 749 4.53 Zone 3 8 304 2 94 480 854 4.72 Solid Zone 2 6 942 2 96 378 713 4.29 Zone 3 6 956 1 95 389 897 4.26 BR 4: (Woodchip/ po a o) Wa e Zone 2 9 261 2 96 303 583 3.61 Zone 3 8 148 2 96 337 605 3.78 Solid Zone 2 9 256 2 97 287 505 3.67 Zone 3 8 359 2 96 296 578 3.39 a OTUs ichness es ima ed by Chao1. 350 Only bac e ial sequences (no a chaeal sequences) we e de ec ed in he bio eac o s. In BR1-BR3, he 351 mic obial communi y was domina ed by P o eobac e ia, Bac e oide es and Ve ucomic obia (Fig. 7). 352 Wi hin he P o eobac e ia, Be ap o eobac e ia we e mos abundan (24-40% ela i e abundance), ollowed 353 by Gammap o eobac e ia (7-26%) and Alphap o eobac e ia (11-28%). In BR4, he mic obial communi y 354 was domina ed by Epsilonp o eobac e ia (15-36%), Bac e oide es (16-29%) and Fi micu es (17-34%) (Fig. 355 7). Amongs he mos abundan gene a, Uliginosibac e ium (up o 11% ela i e abundance), Sul u ospi illum 356 (up o 29%), P e o ella (up o 19%) and Lac obacillus (up o 18%) we e almos exclusi ely de ec ed in BR4, 357 while Rhodobac e (up o 4%), Sphingobium (up o 4%), Rhodo e ax (up o 5%), Pseudomonas (up o 13%), 358 The momonas (up o 6%) and Lu eolibac e (up o 10%) we e almos exclusi ely de ec ed in BR1-BR3 (Fig. 359 20 S3). The gene a Lac obacillus, P e o ella and Sul u ispi illum include known e men e s, some o which can 360 also educe ni a e o ammonium (e.g. K use e al., 2018; Sal e i e al., 2012). The gene a Rhodobac e , 361 Rhodo e ax, Pseudomonas and The momonas include known deni i ie s (e.g. Finne an e al., 2003; 362 Me gae e al., 2003). 363 Fig. 7. Composi ion o he mic obial communi y based on sequence analysis o bac e ial and a chaeal 16S 364 RNA genes om (A) solid ma e ial and (B) wa e samples om woodchip bio eac o s wi h a zone 365 con aining biocha (BR1), Sphagnum sp. moss (BR2), woodchip (BR3) and po a o esidues (BR4). A e age 366 ela i e abundances o 1-2 eplica es pe sample a e shown. Samples we e aken om he op-laye 367 woodchip (zone 3) and he ca bon sou ce ma e ial (zone 2). 368 Func ional p o iles o he bac e ial communi ies we e p edic ed based on 16S RNA gene sequences using 369 PICRUS . I p o ed possible o use a ound 31% o all OTUs and 83% (76-90%) o all sequences o 370 unc ional p edic ion. O e all unc ional p o iles o mic obiological communi ies we e a he simila in he 371 di e en bio eac o s. Selec ed unc ions ela ed o he ni ogen cycle we e assessed in mo e de ail (Fig. 8). 372 21 Func ions ela ed o deni i ica ion (Na G, NapA, Ni K, No B, No C, NosZ) and DNRA (Na G, NapA, 373 N A) we e p edic ed, while unc ions speci ic o ni i ica ion (AmoA, AmoB, AmoC) we e no p edic ed. 374 The memb ane-bound ni a e educ ase Na G was p edic ed in simila ela i e abundance in all bio eac o s, 375 while highe ela i e abundance o he pe iplasmic ni a e educ ase NapA was p edic ed in BR4 han in 376 BR1-BR3 (Fig. 7). The deni i ica ion-associa ed unc ions Ni K, No B, No C and NosZ we e p edic ed wi h 377 highe ela i e abundances o BR1-BR3 han o BR4, while he ni i e educ ase N A (which ca alyses he 378 educ ion o ni i e o ammonia in DNRA) was mo e equen ly p edic ed o BR4 (Fig. 8). 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Deni i ica ion beha io and mic obial communi y spa ial dis ibu ion inside woodchip-based solid-phase deni i ica ion (W-SPD) bio eac o o ni a e-con amina ed wa e ea men . Bio esou . Technol. 249, 869–879. h ps://doi.o g/10.1016/j.bio ech.2017.11.011 - Woodchip bio eac o s emo ed 31-38 g NO 3- -N m -3 d -1 om in ensi e aquacul u e e luen - Addi ional po a o esidues o woodchip ma e ial inc eased 13 % o ni a e emo al a e - The po a o esidue bio eac o hos ed a dis inc ly di e en mic obial communi y Decla a ion o in e es s ☒ The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . ☐The au ho s decla e he ollowing inancial in e es s/pe sonal ela ionships which may be conside ed as po en ial compe ing in e es s: