Performance and microbial features of the partial nitritation-anammox process treating fish canning wastewater with variable salt concentrations

Resea ch a icle Pe o mance and mic obial ea u es o he pa ial ni i a ion- anammox p ocess ea ing fish canning was ewa e wi h a iable sal concen a ions Angeles Val del Rio a , * , And es Pichel a , Nu ia Fe nandez-Gonzalez a , Alba Ped ouso a , And ea F a-V azquez a , Nicolas Mo ales b , Ramon Mendez a , Jose Luis Campos c , Anuska Mosque a-Co al a a Depa men o Chemical Enginee ing, School o Enginee ing, Uni e sidade de San iago de Compos ela, E- 15705 San iago de Compos ela, Spain b Aqualia, Guilla ei WWTP, Camino de la Veiga s/n, E-36720 Tui, Spain c Facul ad de Ingenie ía y Ciencias, Uni e sidad Adol o Ib a~ nez, A da Pad e Hu ado 750, Vi~ na del Ma , E- 2503500, Chile a icle in o A icle his o y: Recei ed 27 Augus 2017 Recei ed in e ised o m 31 Oc obe 2017 Accep ed 4 Decembe 2017 A ailable online 16 Decembe 2017 Keywo ds: Au o ophic Deni ifica ion Fish canning G anule Ni ogen abs ac The pa ial ni i a ion-anammox (PN-AMX) p ocess applied o was ewa e s wi h high NaCl concen a ion was s udied un il now using simula ed media, wi hou conside ing he e ec o o ganic ma e con- cen a ion and he shi in mic obial popula ions. This esea ch wo k p esen s esul s on he applica ion o his p ocess o he ea men o saline indus ial was ewa e . Ob ained esul s indica ed ha he PN- AMX p ocess has he capabili y o eco e i s ini ial ac i i y a e a sudden/acu e sal inhibi ion e en (up o 16 g NaCl/L). Wi h a p og essi e sal concen a ion inc ease o 150 days, he PN-AMX p ocess was able o emo e he 80% o he ni ogen a 7e9 g NaCl/L. The mic obiological da a indica ed ha NaCl and ammonia concen a ions and empe a u e a e impo an ac o s shaping PN-AMX communi ies. Thus, he NOB abundance (Ni ospi a) dec eases wi h he inc ease o he sal concen a ion, while he e o o- phic deni ifie s a e able o ou compe e anammox a e a peak o o ganic ma e in he eeding. ©2017 Else ie L d. All igh s ese ed. 1. In oduc ion The fish canning indus y ep esen s a e y impo an economic sec o in he No hwes o Spain (Galicia), wi h mo e han 65 ac- o ies, loca ed mainly in coas al a eas. As a consequence o his indus ial ac i i y la ge was ewa e olumes a e p oduced, which con ain high solids, o ganic ma e and ni ogen concen a ions. The e o e, hese e fluen s need o be e ficien ly handled p e ious discha ge o a oid p essu es o e he ma ine en i onmen . These e fluen s a e no mally ea ed by means o anae obic ea men echnologies, wi h he main pu pose o educing he chemical ox- ygen demand (COD) concen a ion o he was ewa e and p oduce ene gy as biogas. Ne e heless, he e fluen s coming om he anae obic diges e (AD) con ain high concen a ions o ni ogen, mainly om p o eins, which has o be emo ed in a subsequen s ep o adjus o disposal limi s. Con en ionally, he combined ni ifica ion-deni ifica ion p ocess is applied o he emo al o ni ogen. This p ocess equi es much ene gy o c ea e ae obic condi ions o bac e ial ni ifica ion, as well as he a ailabili y o o ganic ca bon o emo e ni a e by he e o ophic deni i ying o - ganisms (Ka al e al., 2010). I a comple e au o ophic p ocess is applied he o ganic ma e can be sa ed o p oduce mo e ene gy in he AD. Fo his eason, mos o ecen esea ch e o s ha e been ocused on o he al e na i es o ni ogen emo al, such as he combina ion o pa ial ni i a ion and anammox (PN-AMX) p o- cesses. I s applica ion is sui able o was ewa e s eams wi h high ni ogen bu low o ganic ma e concen a ions, like he supe na- an om anae obic diges e s (Lackne e al., 2014). In he PN-AMX p ocess, hal o he ammonium p esen is oxidized ollowing he ni i a ion pa hway, combined wi h he subsequen biological e- ac ion o he p oduced ni i e and he emaining ammonium o p oduce ni ogen gas acco ding o he anammox eac ion. Howe e , *Co esponding au ho . E-mail add esses: [email p o ec ed] (A. Val del Rio), and es.gu ie ez.pichel@ usc.es (A. Pichel), [email p o ec ed] (N. Fe nandez-Gonzalez), alba.ped ouso@ usc.es (A. Ped ouso), [email p o ec ed] (A. F a-V azquez), nicolas.mo ales.pe ei a@ cc.es (N. Mo ales), [email p o ec ed] (R. Mendez), jluis.campos@uai. cl (J.L. Campos), [email p o ec ed] (A. Mosque a-Co al). Con en s lis s a ailable a ScienceDi ec Jou nal o En i onmen al Managemen jou nal homepage: www.else ie .com/loca e/jen man h ps://doi.o g/10.1016/j.jen man.2017.12.007 0301-4797/©2017 Else ie L d. All igh s ese ed. Jou nal o En i onmen al Managemen 208 (2018) 112e121 he p esence o ce ain inhibi o y compounds in he was ewa e may hinde he applica ion o he anammox based p ocesses a indus ial scale (Jin e al., 2012). Fo example, anammox bac e ia a e e y sensi i e o en i onmen al pa ame e s such as salini y (Scaglione e al., 2017), ha is one o he main componen s o he fish canning e fluen s (C is  o ~ ao e al., 2016). The occu ence o high saline concen a ions in fish canning e fluen s, which is mainly caused by a high NaCl concen a ion, may induce sal s ess o he mic oo ganisms in ol ed in he biological ea men s, wi h he subsequen inhibi ion o many enzymes, dec ease in he cell ac i i y and e en ually plasmolysis (Jin e al., 2012). The sal con en is s ongly ela ed o he seasonali y in he fish canning indus y, which may gene a e sudden saline shock loads o unexpec ed a ia ions in he sal concen a ion depending on he changes in he aw ma e ial p ocessed in he ac o y, wi h concen a ion ha a ies om 2 o 35 g NaCl/L (C is  o ~ ao e al., 2016). Al hough, he e a e se e al esea ch wo ks ha epo on he e ec o sal concen a ions on he anammox bac e ia ac i i y, mos o hem show esul s abou ba ch ac i i y es s and/o he con in- uous ope a ion o only he anammox p ocess ed wi h a syn he ic medium (Scaglione e al., 2017). Windey e al. (2005) pe o med he fi s esea ch wo k wi h he PN-AMX p ocess a long e m ope a- ion inc easing he NaCl concen a ion, al hough hey used a syn- he ic medium. Mo e ecen ly, Malo anyy e al. (2015) ackled he adap a ion o one-s ep PN-AMX biomass o inc easing sal con- cen a ions om 0 o 10e15 g NaCl/L in 160 days. In hei s udy, he eeding consis ed in ejec wa e om an anae obic sludge diges e , while he salini y as NaCl was syn he ically added. As he inoculum a ailable o s a -up a PN-AMX p ocess is no mally no adap ed o salini y, an impo an aspec o conside is he possible shi in he mic obial popula ions. Despi e he ad- ancemen s o nex gene a ion sequencing pla o ms in mic obial ecology, he p e ious s udies abou he influence o salini y in he PN-AMX p ocess had paid li le a en ion o his aspec . Mo eo e , only ew s udies ha e analysed he PN-AMX eac o communi ies using his cu ing-edge echnology (Ag awal e al., 2017; Wang e al., 2017b). Fo example, Wang e al. (2017b) s udied he mic o- biological shi in a PN-AMX p ocess wi h he p og essi e inc ease in he sal concen a ion om 0 o 20 g NaCl/L. Howe e , hei esea ch wo k was pe o med wi h a syn he ic medium igno ing he mic oo ganisms ha can be p esen in indus ial e fluen s and he pe u ba ions in composi ion o was ewa e s, such as changes in o ganic ma e compounds. In he p esen esea ch wo k he combined PN-AMX p ocess is applied o he ea men o indus ial saline was ewa e p oduced in a fish canne y. The e ec o sal concen a ions on he pe o - mance o a PN-AMX g anula sludge eac o was e alua ed as: (1) sudden sal shock loads, up o 16 g NaCl/L, and (2) p og essi e adap a ion o concen a ions up o 10 g NaCl/L. The e olu ion o he main mic obial popula ions p esen in he biomass om he eac o was de e mined o p o e he e ec s o salini y and o ganic ma e concen a ion on he PN-AMX pe o mance. 2. Ma e ials and me hods 2.1. Expe imen al se -up A labo a o y sequencing ba ch eac o (SBR) wi h a wo king olume o 1.5 L was used o he expe imen s. The ae a ion sys em consis ed in a diaph agm pump (Labopo N86, KNF) o he ai supply and an ai di use loca ed a he bo om o he eac o . This sys em p o ided good mix u e inside he eac o and he dissol ed oxygen (DO) concen a ion necessa y o he pa ial ni i a ion p ocess. The DO concen a ion was pe iodically measu ed wi h a DO p obe (Hach Lange, model HQ40d Po able Me e ) and was manually egula ed by changing he closing deg ee o an ai al e loca ed in he gas inle conduc ion. The ope a ional cycles we e o 180 min and dis ibu ed as ol- lows: 5 min o ba ch eeding, 160 min o ae a ion, 10 min o se ling and 5 min o e fluen wi hd awal. The hyd aulic e en ion ime (HRT) a ied be ween 0.9 and 1.3 days o all he ope a ional pe iod (Table 1). The indus ial was ewa e was pe iodically collec ed (e e y 1e2 mon hs) a e he AD in ope a ion in a fish canning indus y (Ca oi a, Pon e ed a) and s o ed a 4  C. The indus ial was ewa e was cha ac e ized by a high a iabili y in i s compo- si ion, especially in e ms o NaCl and o ganic ma e concen a- ions (Table 1). The fluc ua ions on he indus ial was ewa e composi ion could no be con olled o p edic ed, and hey we e mainly due o changes in he p ocessed p oduc a he indus ial acili y ( una, mussels, sa dines, e c.), as well as he pe o mance o he anae obic diges e , which wo sened when he indus ial was ewa e con ained high sal concen a ions. 2.2. Ope a ional condi ions Two di e en expe imen s we e assessed o s udy he influence o salini y o e he PN-AMX p ocess ea ing indus ial was ewa e . The fi s expe imen se ed o es he pe o mance o he PN- AMX p ocess a mode a e sal concen a ions and i s pos e io e- co e y a e a shock o salini y ook place. I las ed 150 days and he SBR was ope a ed a labo a o y oom empe a u e (24 ±2  C). The ope a ion was di ided in h ee s ages acco ding o he di e en sal concen a ion o he ed indus ial was ewa e (Table 1). The was ewa e p oduced by he indus y in his pe iod was cha ac- e ized by a mode a e sal concen a ion (be ween 1.7 and 4.3 g Table 1 Ope a ional condi ions and cha ac e is ics o he was ewa e ed o he eac o in he di e en ope a ional s ages. Fi s expe imen Second expe imen S age I S age II S age III S age IV S age V S age VI Days 0e73 74e127 128e155 0e64 65e220 221e445 Tempe a u e (  C) 24.0 ±0.7 23.7 ±1.1 25.6 ±1.3 29.3 ±0.9 30.3 ±1.5 31.0 ±2.1 HRT (d) 1.3 ±0.2 1.1 ±0.1 1.1 ±0.1 0.9 ±0.1 1.0 ±0.1 1.3 ±0.1 DO (mg O 2 /L) 2.7 ±0.5 3.1 ±1.1 4.0 ±0.3 1.4 ±0.5 1.1 ±0.4 0.5e3.5 NaCl (g/L) 3.5 ±0.2 (15.4 ±0.8 a ) 1.7 ±0.3 4.3 ±0.4 3.4e12.2 2.8e8.3 8.6 ±0.9 NH 4 þ (mg N/L) 291 ±51 206 ±22 216 ±15 262 ±29 250 ±44 220 ±35 TOC (mg C/L) 54 ±10 47 ±10 49 ±750±950±945e200 b IC (mg C/L) 345 ±30 285 ±35 349 ±16 324 ±38 299 ±33 350 ±63 PO 4 3 (mg/L) 124 ±16 97 ±21 48 ±12 17e85 96 ±14 13e704 SO 4 2 (mg/L) 82 ±45 76 ±18 156 ±46 89 ±46 111 ±27 0e284 pH 7.8 ±0.1 7.6 ±0.1 7.5 ±0.2 7.6 ±0.2 7.6 ±0.1 7.6 ±0.3 a Punc ual sal peak in days 32e36. b To al o ganic ca bon (TOC) concen a ion e y a iable. A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121 113 NaCl/L) wi h a sudden peak o high salini y (15.4 ±0.8 g NaCl/L) o a sho pe iod o ime (Table 1). The inoculum was PN-AMX g an- ula sludge om a pilo plan o 200 L (ELAN ® p ocess) ea ing he supe na an o a sludge AD in ope a ion in an u ban WWTP (Mo ales e al., 2015). The sys em was inocula ed wi h 7.5 g VSS/L o ELAN ® g anula sludge, cha ac e ized by a specific anammox ac- i i y (SAA) o 0.356 ±0.025 g N/(g VSS$d) a 30  C. This fi s expe imen was s opped due o a ailu e o he AD, and he pos- e io s op o he canne y indus ial ac i i y du ing he summe pe iod ( wo mon hs), which p o oked he una ailabili y o was ewa e . In he second expe imen he a ailable fish canning was ewa e con ained highe salini y han in he p e ious expe imen (>10 g NaCl/L), which could inhibi he anammox p ocess i i we e used di ec ly (based on he esul s o he fi s expe imen ). The e o e, in his second expe imen he p og essi e adap a ion o he PN-AMX p ocess o inc easing sal concen a ions was s udied. The ope a- ional pe iod was di ided in h ee s ages: S age IV co esponded o an inc ease o sal concen a ion om 3.4 o 12.2 g NaCl/L in 41 days; in S age V he adap a ion om 2.8 o 8.3 g NaCl/L was pe - o med in 150 days; finally, in S age VI he sal concen a ion was main ained a 8.6 ±0.9 g NaCl/L, co esponding o he indus ial was ewa e (Table 1). The indus ial was ewa e was mixed wi h he supe na an o an anae obic sludge diges e (Mo ales e al., 2015) in S ages IV and V o ha e inc easing concen a ions o NaCl wi hou he need o dilu e he ni ogen concen a ion o he eeding. The supe na an o he anae obic sludge diges e was used due o i s low NaCl concen a ion (<1 g NaCl/L) and high ammonia concen a ion (app oxima ely 1000 mg NH 4 þ -N/L), as well as he ac ha his was he e fluen ea ed by he biomass used as inoculum. The p opo ion o anae obic sludge diges e supe na an in he eeding was o 35% and 60% a he beginning o S ages IV and V, espec i ely. Then, i was p og essi ely dec eased un il ha ing in he eeding only indus ial was ewa e . The cha ac e is ics o he was ewa e ed o he SBR in each ope a ional pe iod a e p esen ed in Table 1. The inoculum o his second expe imen consis ed in a mix u e o ELAN ® sludge (65% as VSS) wi h sludge om he fi s expe imen (35% as VSS), esul ing in a solids concen a ion o 8.0 g VSS/L wi h a SAA o 0.252 ±0.002 g N/(g VSS$d) a 30  C. In his second expe imen he ope a ional empe a u e was con olled by a he mos a ic ba h and se a 30 ±1  C. 2.3. Analy ical me hods Analy ical de e mina ion o ammonium (NH 4 þ ), ni i e (NO 2  ), ni a e (NO 3  ), pH, o al suspended solids (TSS) and ola ile sus- pended solids (VSS) was ca ied ou acco ding o he s anda d me hods (APHA-AWWA-WPCF, 2005). To al O ganic Ca bon (TOC) con en was de e mined by a Shimadzu analyse (TOC-L, au oma ic sample injec o Shimazdu ASI-L) as he di e ence be ween he To al Ca bon (TC) and he Ino ganic Ca bon (IC) concen a ions. Ca ion and anion concen a ions we e de e mined by ion ch o- ma og aphy wi h an Ad anced Compac IC sys em (861, Me ohm). The mo phology and size dis ibu ion o he g anules we e measu ed by using an image analysis p ocedu e (Tijhuis e al., 1994). Images o he g anula sludge we e aken wi h a digi al came a (Coolsnap, Rope Scien ific Pho ome ics) combined wi h a s e eomic oscope (S emi 2000-C, Zeiss). Fo digi al image analysis he p og amme Image P oPlus ® was used. The quali a i e compo- si ion o he g anules su ace was assessed wi h SEM (scanning elec on mic oscope) echnique, ollowing he p ocedu e o Figue oa e al. (2008). The specific anammox ac i i y (SAA) was de e mined by ba ch assays ollowing he me hodology desc ibed by Dapena-Mo a e al. (2007). The SAA es s we e ca ied ou in closed ials o 25 mL by iplica e a 30  C and 150 pm. The maximum SAA (exp essed as mg N/(g VSS$d)) was de e mined om he slope o he cu e desc ibed by he cumula i e N 2 p o- duc ion wi h he ime and ela ed o he biomass concen a ion in he ials. 2.4. Ra e calcula ions Ammonia and ni i e oxida ion a es (AOR and NOR, espec- i ely) as well as ni ogen emo al a e (NRR) we e es ima ed based on ni ogen balances and he anammox p ocess s oichiome y and exp essed as g N/(L$d), acco ding o Mo ales e al. (2016). The maximum ni ogen emo al pe cen age by a possible he - e o ophic deni ifica ion p ocess (%HD) was de e mined based on o ganic ma e balance acco ding o equa ion (1). %HD ¼TOCin TOCe $0:933 TNin $100 (1) whe e TOC in and TOC e a e he concen a ions o o al o ganic ca bon (as mg C/L) in he influen and in he e fluen , espec i ely. TN in is he o al ni ogen p esen in he eeding (as mg N/L) and 0.933 is he s oichiome ic coe ficien (as g N-NO 3 - /g C) ha ela es ni a e and o ganic ca bon consump ion in he he e o ophic deni ifica ion p ocess conside ing ace ic acid as he sou ce o o ganic ca bon (Gius iniano ich e al., 2018). No e ha pa o he o ganic ma e could be consumed o g ow h and/o ae obically due o he p esence o oxygen, o his eason equa ion (1) con- side s only he “maximum po en ial” alue o he he e o ophic deni ifica ion, no he ac ual. 2.5. Molecula echniques and sequences analysis Biomass samples om he wo inoculums and om he eac o a di e en ope a ional days (fi s expe imen : 30, 65 and 154 days; second expe imen : 55, 183, 386 and 442 days) we e analysed. The samples we e ob ained by ca e ully homogenizing a la ge olume and aking 2 mL aliquo s ha we e s o ed a 20  C un il u he analysis. To al genomic DNA was ex ac ed using he phenole- chlo o o m p o ocol (Alonso-Gu i e ez e al., 2009), quan ified in a Qubi fluo ome e (The mo Fishe Scien ific, Wal ham, MA, USA) and checked o size and in eg i y by s anda d elec opho esis. To p epa e he 16S RNA gene amplicon lib a ies, he V4 hype a iable egion o he 16S RNA bac e ial gene was amplified using he 515F- 806R p ime pai (Gilbe e al., 2010) as desc ibed p e iously by Reguei o e al. (2014). DNA lib a ies we e checked o size quali y and in eg i y using a Bioanalyze (Bioanalyze , Agilen Technolo- gies, San a Cla a, CA, USA). A e de e mining DNA concen a ions by quan i a i e PCR, lib a ies we e pooled in equimola amoun s and sequenced a he genomics uni o he Pa que Cien ífico de Mad id (Spain) on an Illumina MiSeq Sys em (Illumina) using MiSeq Reagen Ki 3 (Illumina). Ini ial sequence quali y-fil e ing and analysis was pe o med as p e iously desc ibed Reguei o e al. (2014). To de e mine he alpha di e si y o he bac e ial communi y, obse ed species, Simpson, Chao1, Shannon and Simpson e enness indices we e calcula ed using 100 a e ac ions o 116,728 sequences in QIIME .1.9.1 (quan i a i e insigh in o mi- c obial ecology) (Capo aso e al., 2010). Be a di e si y, he deg ee o communi y di e en ia ion be ween samples, was measu ed wi h he B ay-Cu is dissimila i ies using log(xþ1) and Hellinge ans- o med ope a ional axonomic uni (OTU) ela i e abundances. Communi y s uc u e a ia ion was isualized using P incipal Componen Analysis ( bPCA) o which he co ela ions be ween mic obial communi y composi ion and ope a ional pa ame e s we e fi ed. Ope a ional pa ame e s we e ans o med as ollows: A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121114 log(xþ1) ans o ma ion was applied o empe a u e, influen ammonium, TOC and sul a e concen a ions whe eas squa ed oo ans o ma ion was applied o dissol ed oxygen concen a ion. The p- alues we e adjus ed o mul iple compa isons using Bon e oni co ec ion wi h 999 pe mu a ions wi h a significance h eshold o 0.05. S a is ical analyses we e pe o med in R (R-Co e-Team, 2016) using he egan package (Oksanen e al., 2016). 3. Resul s and discussion 3.1. Fi s expe imen : salini y shock In he fi s expe imen , un il day 30 o ope a ion (S age I) he ed was ewa e had a sal concen a ion o app oxima ely 3.5 ±0.2 g NaCl/L. The achie ed NRR and AOR had a e age alues o 0.156 ±0.009 g N/(L$d) and 0.103 ±0.005 g N/(L$d), espec i ely (Fig. 1a). The o al ni ogen and ammonium emo al pe cen ages we e app oxima ely 80 and 90%, espec i ely. A e day 31, a new ba ch o eeding collec ed in he indus y p esen ed a highe sal concen a ion. Consequen ly, he sal concen a ion inside he eac o inc eased om 3.5 ±0.2 o 15.4 ±0.8 g NaCl/L in only 35 h (one HRT), and i was main ained in his high alue o 4 ope a- ional days. This sal shock caused a s ong dec ease o e he NRR ( alues close o ze o) while he AOR emained p ac ically cons an (Fig. 1a). The e o e, he ammonium and ni i e concen a ions in he e fluen inc eased o alues abo e 100 mg N/L, being he ni i e concen a ion as high as 200 mg NO 2  -N/L on day 36 (Fig. 1b). These esul s indica e ha he shock o sal inhibi ed anammox bac e ia, while he ammonium oxidizing ac i i y was no a ec ed. A e his b ie episode o sal shock, he sal concen a ion o he was ewa e e u ned o he p e ious alues (app oxima ely 3.3 g NaCl/L), and he ni ogen emo al e ficiency was eco e ed in only 4 days o alues o app oxima ely 80%. In S ages II and III, wi h a sal concen a ion a an a e age alue o 1.7 ±0.3 and 4.3 ±0.4 g NaCl/L, espec i ely, he ni ogen emo al e ficiency was main ained a app oxima ely 80%. The NRR was be ween 0.105 and 0.203 g N/(L$d) and a ied acco ding o he di e en ammonium concen a ion o he ed was ewa e (Fig. 1). Rega ding he concen a ion o he ni ogen species in he e fluen , he ammonium and ni i e concen a ions we e low, while he ni- a e fi ed he expec ed alues o he anammox s oichiome y (Fig.1b). Only in some days o S age II he ni a e concen a ion was highe han he s oichiome ic alue, which co esponded wi h de ec able ni i e oxidizing bac e ia (NOB) ac i i y (NOR in he ange 0.020e0.030 g N/(L$d)). I he p ofiles o NOR a e compa ed o he e olu ion o NaCl concen a ion (Fig. 1b) a sligh NOB ac i i y was de ec ed in S age II, which co ela ed wi h he lowe sal concen a ion, while i was insignifican du ing he es o he ope a ional pe iod. The esul s om his fi s expe imen confi m he sui abili y and s abili y o he PN-AMX p ocess o emo e ni ogen om fish canning was ewa e wi h mode a e sal concen a ions (<5 g NaCl/ L), as well as he good capabili y o quick es o e (4 days) a e a sudden/acu e sal inhibi ion. 3.2. Second expe imen : salini y adap a ion In his second expe imen (S ages IV, V and VI) he possibili y o adap he PN-AMX biomass o ea indus ial was ewa e wi h high sal concen a ions was es ed. In S age IV an inc ease in he sal concen a ion om 3.4 g NaCl/L o 12.2 g NaCl/L was p og es- si ely pe o med du ing 41 days. Howe e , he ime o adap a ion was no enough o anammox bac e ia. The NRR dec eased om alues o 0.189 ±0.035 g N/(L$d) o 0.048 ±0.008 g N/(L$d), while he AOR was main ained a app oxima ely 0.154 ±0.022 g N/(L$d) (Fig. 2a). The loss o anammox ac i i y p o oked he inc ease o ammonium and ni i e concen a ions in he e fluen up o alues o app oxima ely 60 mg NH 4 þ -N/L and 120 mg NO 2  -N/L (Fig. 2b). The o al ni ogen emo al e ficiency dec eased om 80% o 20%. Fo his eason, a he end o S age IV he sal concen a ion was es o ed o alues o app oxima ely 4 g NaCl/L. In S age V a new p og essi e adap a ion was pe o med o inc easing sal concen a ions om 2.8 o 8.3 g NaCl/L h oughou 150 days. Following his s a egy, he PN-AMX p ocess was able o Fig. 1. Da a om he fi s expe imen : (a) Ni ogen emo al a e (NRR, ), ammonium oxida ion a e (AOR, >), ni i e oxida ion a e (NOR, ✕) and NaCl concen a ion (e); (b) Concen a ion o NH 4 þ in he influen (C); NH 4 þ (B), NO 2  (✱) and NO 3  ( ) in he e fluen . Fig. 2. Da a om he second expe imen : (a) Ni ogen emo al a e (NRR, ), ammonium oxida ion a e (AOR, >), ni i e oxida ion a e (NOR, ✕) and NaCl con- cen a ion (e); (b) Concen a ion o NH 4 þ in he influen (C); NH 4 þ (B), NO 2  (✱) and NO 3  ( ) in he e fluen . A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121 115 eco e om he sal inhibi ion episode o p e ious s age. The NRR inc eased om 0.055 g N/(L∙d) on day 70, o a maximum alue o 0.250 g N/(L∙d) on day 216 a a sal concen a ion o 7.7 g NaCl/L (Fig. 2a). In e ms o specific ac i i y, he SAA inc eased om 0.018 g N/g VSS∙d (day 63) up o 0.272 g N/g VSS∙d (day 156). In S age VI he sal concen a ion o he eeding a ied be ween 7 and 10 g NaCl/L, due o he a iabili y o he indus ial was ewa e collec ed. A he beginning, days 221e260, he ni i e concen a ion in he e fluen dec eased om app oxima ely 60 o 25 mg NO 2  -N/L, p obably due o he highe concen a ion o o ganic ma e in he ed was ewa e , wi h TOC concen a ions o 140 ±43 mg C/L, much highe han hose measu ed in p e ious s ages o 40e50 mg C/L (Table 1), which can p omo e he he e o ophic deni ifica ion. On hese days he achie ed NRR was o 0.180 ±0.020 g N/(L$d) o a sal concen a ion o 9.2 g NaCl/L, wi h a o al ni ogen emo al e fi- ciency o 87%. The achie ed NRR alues (S ages V and beginning o S age VI) a e compa able and e en highe han he ones ob ained by Malo anyy e al. (2015) in a mo ing bed biofilm eac o . These au ho s s udied he pe o mance o he PN-AMX p ocess a inc easing sal concen a ions ed wi h anae obic ejec wa e supplemen ed a ificially wi h NaCl. They ob ained, a e an adap- a ion pe iod o 70 days, a NRR o 0.078 g N/L$d a 10 g NaCl/L. Howe e , in compa ison wi h PN-AMX sys ems using saline syn- he ic medium he achie ed NRR alues we e lowe . Fo example, Windey e al. (2005) inc easing he NaCl concen a ion in a o a ing biological con ac o wi h he PN-AMX p ocess ed wi h syn he ic medium achie ed a NRR o 0.609 g N/(L$d) a 30 g NaCl/L a e 172 days o adap a ion. Fu he mo e, in hese p e ious s udies (Malo anyy e al., 2015; Windey e al., 2005), he au ho s obse ed ins abili y o he PN-AMX p ocess due o he inhibi ion o ammonia oxida ion bac e ia (AOB) a high sal concen a ions (>10 g NaCl/L), wi h he subsequen accumula ion o ee ammonia. F om day 260 onwa ds he NRR was a iable and unde 0.1 g N/ (L$d) he mos pa o he ope a ional days. The AOR was mo e s able and i s fluc ua ions we e due o he egula ion o he DO concen a ion equi ed o a oid he accumula ion o ni i e. Fu he mo e, a p og essi e dec ease o he o al ni ogen emo al om 87% o 50% was obse ed. The inc ease o ni i e concen a ion beyond 75 mg NO 2  -N/L om day 270 sugges ed a decline in he anammox ac i i y, confi med by ba ch assays. On day 273 he SAA alue ell o 0.028 g N/(g VSS∙d). As i will be discussed la e , his de e io a ion o he anammox ac i i y migh be caused by he p esence o high concen a ions o o ganic ma e and ela ed o he e o ophic deni ifica ion p ocesses, a ou ing a significan shi in he in ol ed mic obial popula ions. Thus, he esul s ob ained in his second expe imen indica ed ha he anammox bac e ia a e able o ope a e a high sal con- cen a ions (up o 9 g NaCl/L) i enough adap a ion ime is p o ided (app oxima ely 150 days in his esea ch wo k). Howe e , he s a- bili y o he PN-AMX p ocess is limi ed and he p esence o o ganic ma e concen a ions as high as 200 mg C/L (Table 1) in he in- dus ial was ewa e a ec he p ocess nega i ely. 3.3. G anula sludge cha ac e is ics In he fi s expe imen , coinciding wi h he sudden shock o salini y, he concen a ion o biomass inside he eac o dec eased om 6.1 ±0.7 g VSS/L, on day 28, o 3.5 ±0.8 g VSS/L, on day 42. A e wa ds, he biomass con en inside he eac o p og essi ely inc eased, eaching a concen a ion o 7.9 ±0.4 g VSS/L on day 114. The solid concen a ion in he e fluen was lowe han 50 mg VSS/L in S age I. Al hough, alues up o 130 ±7 mg VSS/L we e punc ually ob ained a ound day 65, due o he flo a ion o some g anules. This flo a ion e en was p esumably p o oked by he en apmen o ni ogen gas in he in e nal co e o he g anules. Then, he con- cen a ion o solids in he e fluen was lowe han 25 mg VSS/L in S ages II and III, confi ming he app op ia ed biomass e en ion inside he eac o . The a e age diame e o he g anules inc eased du ing he expe imen al ime om 2.5 ±0.3 (day 7) o 3.8 ±0.2 mm (day 146). In he second expe imen he solid concen a ion inside he eac o was main ained s able wi h an a e age alue o 7.3 ±0.6 g VSS/L. The concen a ion o solids in he e fluen was o 100 ±30 mg VSS/L. Al hough, alues up o 200 ±17 mg VSS/L we e punc ually ob ained on day 273, again due o g anule flo a ion episodes. The a e age diame e o g anules in his expe imen was o 3.3 ±0.4 mm. To unde s and he impo ance o floa a ion e en s SAA es s we e pe o med o floa ing and se ling g anules on day 273, esul ing in alues o 0.061 ±0.011 and 0.028 ±0.001 g N/(g VSS$d), espec i ely. These esul s indica e ha he floa ing g anules had wo- old he ac i i y o he se ling ones, and emphasize he impo ance o a oiding he wash ou o his ac ion o he biomass. Campos e al. (2017) s a ed ha he floa a ion o anammox g anula biomass occu s when he ni ogen gas emains en apped inside he g anule due o he o e loading o he sys em. These au ho s s a ed ha he accumula ion o ni i e in he liquid phase and he inc ease o he g anules size a e ac o s ha p omo e he g anules floa a ion. As bo h ac o s occu ed in he p esen esea ch wo k, hey p obably we e esponsibly o floa a ion e en s o he g anula biomass. Ano he e en ha ook place wi h he biomass in he second expe imen was he appea ance o a whi ish laye o p ecipi a es on he su ace o some g anules (Supplemen a y ma e ial, Figu e S1). This ac was obse ed o he fi s ime on day 292. An elemen al analysis o he g anules su ace using SEM was pe o med in zones wi h and wi hou such p ecipi a es. The esul s ob ained we e only quali a i e, bu indica ed ha he main di e ence be ween bo h zones was a high p esence o elemen al sulphu in he p ecipi a es zone. The o ma ion o hese su ace p ecipi a es may sugges a pa ial explana ion o he obse ed loss in he anammox ac i i y, due o he appea ance o subs a e ans e limi a ions be ween he liquid media and he inne laye s o he g anules, whe e anammox a e loca ed. Dapena-Mo a e al. (2010) obse ed he o ma ion o p ecipi a es o e g anula anammox biomass when ea ing high saline was ewa e up o 30 g NaCl/L, being Ca 3 (PO 4 ) 2 he main compound jus i ying hese p ecipi a es. 3.4. Bac e ial communi y o PN-AMX eac o To in es iga e he bac e ial di e si y in he PN-AMX, 16S RNA bac e ial gene lib a ies we e cons uc ed and sequenced a di e en imes h oughou he en i e s udy. Mo e han 1.3 M high- quali y sequences (148,905 ±19,039 pe sample) we e ob ained and clus e ed in o OTUs a 3% cu o (Supplemen a y ma e ial, Table S1). The bac e ial di e si y and ichness emained s able (Table 2) despi e he changes on ope a ional condi ions, indica ing he p esence o a salini y adap ed communi y in he eac o . P e- ious s udies in simila sys ems obse ed a di e si y loss wi h inc easing salini y le els due o he selec i e p essu e o salini y in non-adap ed communi ies (Wang e al., 2017a); bu also a di e si y eco e y once he communi y became adap ed (Wang e al., 2017b). The PN-AMX eac o mic obial communi y is e y complex (es i- ma ed species: 1069 ±56; Simpson di e si y, he highe index he mo e di e se communi y: 0.97 ±0.01). Howe e , he communi y is ma kedly une en (Simpson e enness, he highe he index he mo e e en communi y: 0.034 ±0.009) indica ing he low e enness o he communi y, ha was domina ed by ew o ganisms and main ained nume ous low abundan bac e ia ( a e bac e ia). A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121116 The bac e ial communi y simila i y a OTU le el ac oss di e en ime poin s was e alua ed using B ay-Cu is dissimila i ies (Fig. 3; Supplemen a y ma e ial, Figu e S2). The esul s indica ed ha communi y s uc u e was dynamic h oughou ime, shi ing du - ing he di e en s ages. Despi e belonging o di e en expe imen s, samples om bo h inoculums and he eac o be o e su e ing any NaCl peak (S age I a day 30) we e simila , and we e clea ly sepa- a ed om he communi ies du ing he mid-s ages. Bu he mos dis inc communi ies we e hose om he la e days o he las expe imen al s age when sus ained salini y concen a ions we e he highes . The bPCA analysis confi med ha hese changes o he communi y s uc u e we e co ela ed wi h a ia ions in he influen concen a ion o NaCl and NH 4 þ and empe a u e o ope - a ion (Fig. 3; Supplemen a y ma e ial, Figu e S3). All hese h ee pa ame e s a e well known o influence anammox and ni i ying mic oo ganisms ac i i ies (Ag awal e al., 2017; Gonzalez-Ma inez e al., 2015; Lo i e al., 2015; Ma e al., 2016; Wang e al., 2017a, 2017b). The bac e ia axonomic di e si y was unequally dis ibu ed ac oss 26 phyla, whe e he 10 mos abundan axa ep esen ed o e he 90% o he bac e ial communi y (Fig. 4a). The mic obial com- muni y was domina ed by o ganisms o he P o eobac e ia phylum (a e age ela i e abundance: 52.6 ±11.5%), especially om he Be a (20.2 ±1.5%), Alpha (13.3 ±4.7%) and Gammap o eobac e ia (10.5 ±3.9%) classes. O he majo phyla included Fi micu es (9.3 ±5.2%), Bac e oide es (9.0 ±3.4%), Planc omyces (6.2 ±3.9%), Ac inobac e ia (3.6 ±1.3%) and Chlo obi (2.1 ±1.3%). These axa a e commonly ound in pa ial ni i a ion and/o anammox eac o s (Ag awal e al., 2017; Dos a e al., 2015; Gonzalez-Ma inez e al., 2015; Wang e al., 2017b). The low ela i e abundance o Planc o- myce es despi e he high anammox ac i i y is in acco dance wi h p e ious obse a ions (Dos a e al., 2015; Gonzalez-Ma inez e al., 2015). Chlo oflexi, known o co-exis s wi h anammox bac e ia (Yamagishi e al., 2013; Yamamo o e al., 2011), is hough o sca enge o ganic ma e om he dead biomass (Kindaichi e al., 2012) and suppo g anules o ma ion due o i s filamen ous cha ac e is ics (Ni e al., 2011). The la ges ac ion o he communi y was cha ac e ized by he e o ophic bac e ia. Many o he p edominan amilies (Fig. 4b) we e he e o ophic bac e ia such as Rhodocyclaceae, Rhodobac e - aceae,Comamonadaceae, Xan homonadaceae, Bu kholde iaceae, Igna ibac e iaceae,Hyphomic obiaceae o Fla obac e iaceae. P e ious analyses o anammox eac o s ha e al eady shown ha he e o ophic bac e ia ep esen a la ge ac ion o he communi y (Ag awal e al., 2017; Cos a e al., 2014; Dos a e al., 2015; Ga cia Cos as e al., 2012; Kindaichi e al., 2007; Langone e al., 2014; Lau eni e al., 2015; Ni e al., 2012; Pe sson e al., 2017). Spe h e al. (2016) hypo hesized ha he exis ence o a iable mac o- and mic o-en i onmen s allows he coexis ence o such la ge di- e si y o he e o ophic bac e ia in he eac o s. Many o hese amilies also con ain known comple e o pa ial deni ifie s and deni ifica ion in e media e educe s (Liu e al., 2012; Ni e al., 2011). Many o he majo OTUs (a e age ela i e abundance >0.5%) seemed o be influenced by he ammonium, salini y and TOC concen a ions o he eeding and he empe a u e o he ope a ion (Fig. 5a; o he influence o pa ame e s a phyla le el see Supple- men a y ma e ial Figu e S3). High salini y and empe a u e con- di ions we e co ela ed wi h he p esence o he e o ophic deni ifie s Thaue a,Pa acoccus,Thio ix when TOC was high. Inc easing Thaue a abundances wi h salini y has been al eady e- po ed in a simul aneous ni ifica ion, deni ifica ion and o ganic ma e emo al p ocess (Wang e al., 2017a). High le els o ammonium and TOC a o ed many he e o ophs like Cohnella, Bac e oide es,Fi micu es,Chi inophaga. Anammox bac e ia abun- dances we e no co ela ed by any o he pa ame e s s udied. Two anammox bac e ia we e ound wi hin he majo genus (Supplemen a y ma e ial, Figu e S4): Candida us B ocadia (4.6 ±3.0) and Candida us Scalindua (1.2 ±0.8), bo h wi h simila empo al endencies. P e ious s udies de e mined ha C. B ocadia p edomina e in eac o communi ies unde high ni i e concen- a ions and COD/N a ios (Jenni e al., 2014; Lau eni e al., 2015). Ni ospi a was he mos abundan NOB de ec ed, al hough he gene a a e age ela i e abundances we e lowe han 0.6%. Ni o- spi a has been p e iously epo ed as he main NOB o a PN-AMX sys em unde high salini y condi ions (Wang e al., 2017b). Ni o- bac e , and some AOBs (Ni osospi a,Ni oso ib io and Ni o- sococcus) we e also de ec ed wi hin he a e bac e ia (<0.01%). Table 2 Bac e ial alpha di e si y, indices o ichness, di e si y and e enness. Fi s expe imen Second expe imen S age I S age III S age IV S age V S age IV Day 0 30 65 154 0 55 183 386 442 Obse ed Species 1150 1088 1124 1007 1039 1067 972 1097 1074 Simpson di e si y 0.967 0.971 0.980 0.960 0.967 0.983 0.966 0.974 0.973 Shannon 6.626 6.508 6.882 6.094 6.389 6.909 6.195 6.543 6.440 Chao1 1488 1335 1296 1245 1279 1313 1200 1370 1369 E enness 0.026 0.032 0.044 0.025 0.030 0.055 0.031 0.035 0.034 Fig. 3. Bac e ial communi y s uc u e a OTU le el and i s ela ionship wi h he ope a ional pa ame e s: empe a u e (T), sal (NaCl) and ammonia (NH 4 þ ) concen a- ions. The bPCA analysis o he B ay-Cu is dissimila i ies and he ope a ional pa- ame e s ha a e significan ly co ela ed wi h he communi y s uc u e (p- alue <0.05). The % o communi y a iance explained by each axis is indica ed. G oups o samples wi h dissimila i ies below 0.4 a e indica ed wi h g ey dashed ci cles. Labels indica e ope a ional day. Opened symbols indica e he fi s expe imen samples: Inoculum 1 (,), S age I (B), S age III ( ▵ ). Closed symbols indica e second expe imen samples: Inoculum 2 (-), S age IV (C), S age V (A), S age IV (:). A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121 117 P e ious s udies ha e epo ed low ac ions o AOBs and NOBs in PN-AMX communi ies (Ag awal e al., 2017; Alms and e al., 2014). In he p esen esea ch wo k howe e no AOBs we e de ec ed among he abundan bac e ia. One plausible explana ion could be he p esence o Ammonia Oxidizing A chaea (AOA). Un- o una ely, A chaea was no explo ed in his s udy, al hough we ha e obse ed he en ichmen o Ni osopumilus in he A chaea ac ion o o he anammox eac o s in ou lab (unpublished da a). AOA g oup has been de ec ed as he dominan ni i ying commu- ni y in a ma ine aqua ium biofil e and appea s o be widesp ead in WWTPs (Junie e al., 2010) and ha AOA can ou compe e AOB as hey a e less sensi i e o salini y (Wang e al., 2017a). Tempo al endencies o he majo gene a o anammox bac e ia (C. B ocadia,C.Scalindua), (pa ial) deni ifie s (Thaue a,Pa acoccus, Bu kholde ia,Comamonas,Thiobacillus and Pedobac e ) and NOB (Ni ospi a) a e indica ed in Fig. 5b (see Supplemen a y ma e ial Figu e S4 o indi idual ends). P esence o NOB was expec ed when NOR ac i i y was highe and NaCl concen a ions we e ela i ely low. In he fi s expe imen , Ni ospi a emained a low ela i e abundances o S age I. A e wa ds, Ni ospi a abundance p esumably inc eased du ing S age II (no 16S da a a ailable) because he ise o NOR. This ac can jus i y he p esence o Ni ospi a a day 154 (S age III). As a esul , he ela i e abundances o Ni ospi a du ing S age III we e highe han a S age I, bu p obably lowe han du ing S age II. In he second expe imen , Ni ospi a was low in all samples excep a day 55 in S age IV. In his case, Ni ospi a p obably g ew du ing days 20e23, when NOR inc eased and he salini y was s ill mode a ed, and al hough i was likely inhibi ed by he sal inc emen o days 30e50 he ela i e abundances measu ed a day 55 we e s ill eflec ing ha inc e- men . Then, when NaCl was kep o e 6 g/L, Ni ospi a d opped in abundance, in ag eemen wi h he NOR da a; and emained as a e bac e ia as o he NOBs (Ni obac e ). Anammox bac e ia inc eased almos s eadily om S age I o S age III. The small d op obse ed be ween days 30 and 65 was p obably ela ed wi h he inhibi ion du ing he sal peak. The da a du ing S age IV and V showed an en ichmen o anammox. How- e e , anammox dec eased du ing S age VI when he NRR d opped. In his s age, he deni i ying bac e ia no iceably inc eased hei impo ance in he communi y. The empo al ends o anammox and deni i ying bac e ia a e opposi e, p obably eflec ing he compe i ion o esou ces. Fig. 4. Taxonomic p ofile o he inoculum and he PN-AMX eac o bac e ial communi y a di e en imes. (a) Mos abundan phyla. Fo P o eobac e ia phylum, he di e en classes a e shown. (b) Mos abundan amilies. Sample labels indica e: inoculum o fi s expe imen (In.1) o second expe imen (In.2) o he eac o S ages (S .I-VI) ollowed by he ope a ional day. A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121118 3.5. Ni i e oxidizing bac e ia (NOB) ac i i y The p esence o significan NOB ac i i y is one o he mos common p oblems ha educe he ni ogen emo al e ficiency in a PN-AMX sys em (Lackne e al., 2014). Among he di e en pa- ame e s ha can be used o supp ess he NOB ac i i y, se e al s udies ha e confi med he sal s ess as a good s a egy, because NOB a e mo e sensi i e han AOB o saline condi ions (She e al., 2016). The e o e, he ea men o saline e fluen s by he applica- ion o PN-AMX p ocess is conside ed as a sui able op ion (Liu e al., 2008). Also his sal s ess may be used as a echnical app oach o inhibi he NOB ac i i y du ing he s a -up o a PN-AMX p ocess o indus ial saline was ewa e s, as o he au ho s ound wo king wi h syn he ic mine al medium (Zhang e al., 2010). Ne e heless, ca e mus be aken in his case and a sal inc easing s a egy long enough o ensu e he adap a ion o anammox biomass o high sal condi ions mus be applied. The esul s ob ained in his esea ch wo k showed ha he NOR was only significan du ing he fi s days o bo h expe imen s and in S age II (Fig. 1a and 1b). In he fi s expe imen he NOR dec eased om 0.058 ±0.002 g N/(L$d) (day 8) o ze o, in only 5 days wi h a sal concen a ion o 3.6 g NaCl/L. Then, in S age II a a sal concen a ion o 1.7 g NaCl/L, he NOR had an a e age alue o 0.014 ±0.010 g N/(L$d). The inc ease o he NOR in S age II can be co ela ed wi h he dec ease o he sal concen a ion (Fig. 1a). In he second expe imen he NOR dec eased om 0.032 ±0.001 g N/ (L$d) (day 27) o ze o in 15 days. In his pe iod, he sal concen- a ion inc eased om 3.4 o 12.2 g NaCl/L. These esul s indica e ha he saline condi ions helped o main ain a low NOB ac i i y inside he eac o . Howe e , he sal concen a ions es ed we e no enough o comple ely wash ou he NOB. Ni ospi a, as well as o he NOB bac e ia, could subsis as pa o he a e membe s o he bac e ia communi y when he salini y was high. I he condi ions become a ou able (low sal concen- a ion) hey can de elop significan ac i i y in only ew days. The numbe o epo s showing he g ow h o a e membe s o he communi y o become pa o abundan ac ion wi h he adequa e condi ions is inc easing. I has been hypo hesized ha he a e biosphe e se es as a ese oi o di e si y ha inc eases he plas ici y and adap abili y o he communi ies (Shade and Gilbe , 2015). This phenomenon in an en i onmen such as he PN-AMX can be an incon enien as NOBs a e no o ally wash-ou o he sys em. 3.6. Influence o o ganic ma e and he e o ophic deni ifica ion The p ocess o he e o ophic deni ifica ion is known o occu simul aneously wi h he PN-AMX p ocess when biodeg adable o ganic ma e is a ailable. This he e o ophic deni ifica ion a- cili a es he emo al o he ni a e p oduced due o he anammox bac e ia ac i i y and, consequen ly, ni ogen emo al e ficiencies highe han 89% can be achie ed (Gius iniano ich e al., 2016). Howe e , he de elopmen o he e o ophic deni ifica ion migh b ing a shi in he mic obial popula ions beha iou and compe e wi h anammox bac e ia o he ni i e (Jenni e al., 2014). In he fi s expe imen and un il day 220 o he second one, he o al o ganic ca bon o ni ogen (TOC/N) a io in he eeding was lowe han 0.25 g TOC/g N. The es ima ed maximum ni ogen emo al pe cen age due o a possible he e o ophic deni ifica ion p ocess was calcula ed o each ope a ional pe iod wi h Equa ion (1) and esul ed o be lowe han 12% (Fig. 6). Fu he mo e, he mola a io o ni a e-N p oduc ion o ammonium-N consump ion was close o he s oichiome y alue o anammox p ocess (0.11 g NO 3  -N p oduced /g NH 4 þ -N consumed ), wi h a e age alues o 0.11 ±0.04, 0.18 ±0.06, 0.16 ±0.02 and 0.15 ±0.05 g NO 3  -N p oduced /g NH 4 þ - N consumed o S ages I, II, III and IV, espec i ely. Only in S age V his a io was lowe han he expec ed (a e age alue o 0.05 ±0.01 g NO 3  -N p oduced /g NH 4 þ -N consumed ), which could indica e a possible ni a e consump ion by he deni ifica ion p ocess. In ac , some Fig. 5. a) The bPCA analysis o majo OTUs ela i e abundances and he ope a ional pa ame e s (p- alue <0.05). The % o communi y a iance explained by each axis is indica ed. T: empe a u e; Ana: Anae olineaceae, Bac: Bac e oide es, Be : Be ap o eo- bac e ia, B o: Candida us B ocadia, Caa: Candida us Amoebophilus asia icus, Cam: Campylobac e ales, Chi: Chi inophaga soli, Cmm: Comamonadaceae, Coh: Cohnella soli, Dk : Dokdonella ugi i a, Dok: Dokdonella sp., Fi : Fi micu es, Fla: Fla obac e iaceae, Ign: Igna ibac e ium, Lew: Lewinella sp., Lwm: Lewinella ma ina, Meg: Megasphae a hominis, Pa : Pa acoccus sp., Psu: Pa acoccus sul u oxidans, Rho: Rhodociclaceae, Rhp: Rhodocyclus pu pu eous, Sca: Candida us Scalindua b odae, Tha: Thaue a sp., Thl: Thaue a linaloo- len is, Th : Thaue a e penica, Thx: Thio ix sp., Xan: Xan homonadaceae. (b) Tempo al changes o he majo anammox bac e ia, (pa ial) deni ifie s and NOB. The NaCl ange o each day is indica ed in he g ey ho izon al ba . No ice he opposi e ends be ween anammox bac e ia and deni ifie s. Fo sample label desc ip ion see Fig. 4 cap ion. A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121 119 pu a i e au o ophic deni i ying bac e ia a e p esen du ing hose s ages. The e o e, in hese ope a ional s ages he PN-AMX p ocess was he main mechanism o ni ogen emo al and anammox bac- e ia ou compe ed deni i ying mic oo ganisms. In he second expe imen , om day 221 on (S age VI), he TOC/N a io in he eeding was a iable and in some ope a ional pe iods highe han 0.30 g TOC/g N, which p o oked a compe i ion be- ween anammox and deni i ying bac e ia. Be ween days 221 and 260 a high o ganic ma e peak was de ec ed in he eeding due o a new indus ial ba ch o was ewa e , wi h a TOC/N a io o 0.66 ±0.07 g TOC/g N. On hese days, he es ima ed maximum ni ogen emo al pe cen age due o a possible deni ifica ion p o- cess was calcula ed o be as high as 53% (Fig. 6). Fu he mo e, he a io o ni a e-N p oduc ion o ammonium-N consump ion was e y low (<0.006 g NO 3  -N p oduced /g NH 4 þ -N consumed ), which in- dica es a possible consump ion o ni a e by he e o ophic deni- ifica ion. In his pe iod (days 221e260) he o al ni ogen emo al e ficiency had he highe alues o he expe imen (app oxima ely 87%). Thus, he imp o emen in he ni ogen emo al in his case was no due o an imp o emen in he anammox ac i i y, bu o a ise in he he e o ophic deni ifica ion p ocess d i en by he o ganic ma e con en . When he TOC/N a io dec eased in he ollowing days, he o al ni ogen emo al e ficiency was no main ained ollowing a dec easing end (Fig. 6), despi e ha ano he ele an peak o o ganic ma e was de ec ed be ween days 351e375. These obse a ions can be a ibu ed o he p oli - e a ion o he e o ophic mic oo ganisms be ween days 221e260 and 351e375. He e o ophic bac e ia can displace anammox compe ing o ni i e due o hei high g ow h a es (Liang e al., 2014; Zhang e al., 2012). The changes in he TOC/N a io p o- duced a la ge shi in he communi y composi ion du ing S age VI by s imula ing he g ow h o he e o ophic deni ifie s (i.e: Thaue a,Pa acoccus) and he d op o anammox bac e ia. O e all, he o ganic ma e con en can ac as a supplemen a y aid o he ni ogen emo al p ocess, as p e iously epo ed in li e a u e (Gius iniano ich e al., 2016; Jenni e al., 2014). Howe e , he occu ence o sudden inc eases in he TOC/N a io o he ed was ewa e may end up shi ing he compe i ion be ween he e o- ophic deni ifie s and anammox bac e ia. 4. Conclusions The s udy o he PN-AMX p ocess wi h indus ial saline was e- wa e indica ed ha a e a sho sal shock (4 days a 16 g NaCl/L) he anammox bac e ia can quick es o e hei ac i i y in ew days. Wi h enough adap a ion ime (150 days) he PN-AMX p ocess showed a good o al ni ogen emo al e ficiency (80%) and NRR (0.2 g N/L$d) a 7e9 g NaCl/L. Howe e , he p esence o o ganic ma e in he indus ial was ewa e , a concen a ions as high as 200 mg TOC/L, des abilized he p ocess. The mic obiological da a confi med ha high NaCl concen a ions dec eased he NOB abundance, while he p esence o o ganic ma e a ou s ha he e o ophs displace anammox bac e ia. Acknowledgemen s This wo k was suppo ed by he Spanish Go e nmen h ough GRANDSEA (CTM2014-55397-JIN) and FISHPOL (CTQ2014-55021- R) p ojec s co- unded by FEDER, and he Chilean Go e nmen (CONICYT/FONDAP/15130015). The au ho s om he USC belong o CRETUS (AGRUP2015/02) and he Galician Compe i i e Resea ch G oup (GRC 2013-032), p og ams co- unded by FEDER. Appendix A. Supplemen a y da a Supplemen a y da a ela ed o his a icle can be ound a h ps://doi.o g/10.1016/j.jen man.2017.12.007. Re e ences Ag awal, S., Ka s , S.M., Gilbe , E.M., Ho n, H., Nielsen, P.H., Lackne , S., 2017. 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Fig. 6. Pe cen ages o ni ogen emo al (in o e lapping ba s): o al ni ogen (TN, ,) and maximum possible he e o ophic deni ifica ion (HD, ). TOC/N a io in he indus ial eeding (C). A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121120