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.
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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
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A. Val del Rio e al. / Jou nal o En i onmen al Managemen 208 (2018) 112e121120