Science o he To al En i onmen 912 (2024) 169349
A ailable online 15 Decembe 2023
0048-9697/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC license (h p://c ea i ecommons.o g/licenses/by-
nc/4.0/).
En i onmen al p o eomics as a use ul me hodology o ea ly-s age
de ec ion o s ess in anammox enginee ed sys ems
Víc o Guzm´
an-Fie o
a
, Albe o Dieguez-Seoane
b
, Ma lene Roeckel
a
, Juan M. Lema
b
,
Alba T ueba-San iso
b
,
*
a
Depa men o Chemical Enginee ing, Facul y o Enginee ing, Uni e si y o Concepci´
on, Concepci´
on, Chile
b
CRETUS, Depa men o Chemical Enginee ing, Uni e si y o San iago de Compos ela, Campus Vida, San iago de Compos ela, Galicia, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•Anammox s ess was de ec able a e
only 28 h o exposu e o coppe .
•S ess p o eins we e o e exp essed.
•Polysaccha ide-biosyn hesis p o ein and
algina e expo e e ealed highe EPS
p oduc ion.
•The p esen ed wo k low can be used o
s udying o he me als o con aminan s
e ec s.
•En i onmen al p o eomics can assess
he ea abili y o indus ial e luen s.
ARTICLE INFO
Edi o : Damia Ba celo
Keywo ds:
Anammox p ocess
Indus ial e luen s
Ni ogen emo al
P o eomics
S ess-moni o ing
Was ewa e ea abili y
ABSTRACT
Anammox bac e ia a e widely applied wo ldwide o deni i ica ion o u ban was ewa e . Di e en ly, hei
applica ion in he case o indus ial e luen s has been mo e limi ed. Those equen ly p esen high loads o
con aminan s, demanding an indi idual e alua ion o hei ea abili y by anammox echnologies. Bio eac o s
se ing up and eco e y a e con aminan s-de i ed pe u ba ions a e slow. Also, oxici y is equen ly no acu e
bu cumula i e, which causes nega i e mac oscopic e ec s o appea only a e medium o long- e m ope a ions.
All hese pa icula i ies lead o ele an economic and ime losses. We hypo hesized ha con aminan s cause
changes a anammox p o eome le el be o e pe u ba ions in he enginee ed sys ems a e de ec able by mac o-
scopic analyses. In his s udy, we explo ed he use ulness o sho -ba ch es s combined wi h en i onmen al
p o eomics o he ea ly de ec ion o hose changes. Coppe was used as a model o s esso con aminan , and
anammox g anules we e exposed o inc easing coppe concen a ions including p e iously epo ed IC
50
alues.
The p o eomic esul s e ealed ha speci ic anammox p o eins in ol ed in s ess esponse (bac e io e i in,
uni e sal s ess p o ein, o supe oxide dismu ase) we e o e exp essed in as sho a ime as 28 h a he highe
* Co esponding au ho a : CRETUS, Depa men o Chemical Enginee ing, Uni e si y o San iago de Compos ela, Campus Vida, 15782 San iago de Compos ela,
Galicia, Spain.
E-mail add ess: [email p o ec ed] (A. T ueba-San iso).
Con en s lis s a ailable a ScienceDi ec
Science o he To al En i onmen
jou nal homepage: www.else ie .com/loca e/sci o en
h ps://doi.o g/10.1016/j.sci o en .2023.169349
Recei ed 15 Sep embe 2023; Recei ed in e ised o m 11 Decembe 2023; Accep ed 11 Decembe 2023
Science o he To al En i onmen 912 (2024) 169349
2
coppe concen a ions. Consequen ly, EPS p oduc ion was also inc eased, as indica ed by he algina e expo
amily p o ein, polysaccha ide biosyn hesis p o ein, and sul o ans e ase inc eased exp ession. The desc ibed
wo k low can be applied o de ec ea ly-s age s ess bioma ke s o he nega i e e ec o o he me als, o ganics, o
e en changes in physical-chemical pa ame e s such as pH o empe a u e on anammox-enginee ed sys ems. On
an indus ial le el, i can be o g ea alue o decision-making, especially be o e dealing wi h new e luen s on
acili ies, de i ing impo an economic and ime sa ings.
1. In oduc ion
Anae obic ammonium oxidizing bac e ia (anammox) a e chemo-
li ho ophs ha gain hei ene gy o g ow h om he anae obic
oxida ion o ammonium, using ni i e as an elec on accep o o p oduce
N
2
(Kuenen, 2008; Ka al e al., 2011). In na u e, anammox is es ima ed
o con ibu e up o 50 % o he ni ogen in he global ai (Ka al e al.,
2012; Ka al e al., 2013). These bac e ia ha e been ubiqui ously
de ec ed in anoxic en i onmen s whe e ni ogen compounds a e
deg aded, and besides hei biogeochemical and ecological ele ance,
hey ha e also been applied wo ldwide in enginee ed sys ems o u ban
was ewa e deni i ica ion (WWTPs) (Kuenen, 2008). Anammox ech-
nology has lowe oxygen and ca bon sou ce demand, as well as lowe
biomass p oduc ion and g eenhouse gas emissions when compa ed o
con en ional sys ems (Ren e al., 2020). I ep esen s a clean and cos -
e ec i e solu ion o biological deni i ica ion p ocesses in line wi h
he Sus ainable De elopmen Goals 6, 11, and 13 om he Uni ed Na-
ions (Uni ed Na ions, 2015).
Anammox bac e ia a e ed-colo ed, due o hei high con en o cy-
och omes (Ka al e al., 2013) and hei key ni ogen ans o ma ions
occu inside an in acellula o ganelle, named anammoxosome which
makes hei cell a chi ec u e unique (Ka al e al., 2012). Addi ionally,
anammox can g ow on g anules (i.e., sel -agg ega ed bio ilms) by p o-
ducing ex acellula polyme ic subs ances (EPS) in which he cells a e
embedded (Ka al e al., 2012). In ad anced WWTPs, ope a ional pa-
ame e s a e op imized o a o his g anula ion ha imp o es he
se ling eloci y and biomass e en ion (S ous e al., 1998; Zhang e al.,
2015; Mo e e al., 2014). Anammoxosome enzymes ely on coppe , i on,
and molybdenum as co ac o s (Reimann e al., 2015), and he e o e
me als, as well as ammonium and ni i e, should espass he complex
mix u e o p o eins, nucleic acids, phospholipids, humic subs ances,
polysaccha ides, and in e cellula polyme s ha o m he EPS (Mo e
e al., 2014) o each he ca aly ic uni s.
To da e, he applica ion o a one-s age ni i a ion/anammox p ocess
o indus ial was ewa e has been mo e limi ed, when compa ed o
u ban wa e s. Ye , a ew epo s in he li e a u e documen ed success ul
o explo a i e applica ions wi h di e en e luen s (land ill leacha e,
monosodium glu ama e was ewa e , pigge y was ewa e , e c.) in
di e en ypes o bio eac o s including sequencing ba ch eac o ,
luidized-bed, ixed-bed, up- low anae obic sludge bed o memb ane
bio eac o s (Ren e al., 2022; Li e al., 2018a, 2018b). The main hin-
d ance o he p ac ical applica ion o anammox echnologies elies on
hei slow se ing up (in he ange o mon hs) due o he obse ed long
doubling imes (15–30 days) and low speci ic g ow h a es (Ka al e al.,
2012; Reino e al., 2018) o hese bac e ia, making he eco e y a e
pe u ba ions such as hose de i ed om he p esence o con aminan s
also e y slow.
Ni ogen- ich indus ial was ewa e , such as hose whe e he
ni i a ion-anammox p ocess is easible equen ly con ains high loads o
con aminan s, including o maldehyde, pha maceu icals, o hea y
me als ha can a ec he pe o mance o hese mic oo ganisms. Mos o
he p e ious esea ch e o s ocused on he mac oscopic nega i e e ec s
o pollu an s on he anammox pe o mance ( emo al a es) o se ling
cha ac e is ics (g anules diame e ) (Yang e al., 2013; Li e al., 2015;
Zhang e al., 2016). F equen ly, oxici y is no acu e bu cumula i e, and
inhibi ion akes place in he medium o long e m. The e o e, acu e
oxici y es s do no an icipa e hose pe u ba ions and long- e m
expe imen s a e money and ime-consuming. Taking all in o conside -
a ion i is ele an o explo e as e al e na i es o e alua e he easi-
bili y o anammox echnologies o ea new speci ic was ewa e . The
au ho s hypo hesized ha molecula changes ha occu in he cells
be o e he dec ease in he ac i i y a e de ec able by con en ional
physical-chemical analyses, and be o e he sys em is des abilized.
Consequen ly, i is o in e es o ind a sui able de ec ion me hodology
and o iden i y bioma ke s o ea ly-s age s ess.
En i onmen al p o eomics is an umb ella e m o a se o molecula
echniques ha allow he iden i ica ion o he p o eins being exp essed
by a mixed mic obial communi y a a speci ic momen (Zhang e al.,
2013). They ha e been inc easingly applied o he s udy o mic obiomes
in ol ed in bio echnological p ocesses p o ing o be success ul in
de ec ing speci ic me abolic changes in highly complex communi ies
(Qui on-Tapia e al., 2023) o con i ming he biodeg ada ion o speci ic
pollu an s (Kennes-Veiga e al., 2022; Poulsen e al., 2023). Fo ins ance,
Wang e al. (2021) de ec ed a educ ion in he exp ession o key p o eins
om Ca. B ocadia ni ogen and ca bohyd a e me abolism du ing apid
empe a u e d ops a ec ing he pe o mance o a g anula anammox
eac o .
Al hough coppe (Cu) is essen ial o cell me abolism, excessi e
concen a ions a e known o cause mic obial inhibi ion nega i ely
a ec ing mac oscopic pa ame e s such as ni ogen emo al a es (NRR),
o speci ic anammox ac i i y (SAA) as i has been well e lec ed in he
li e a u e (Madei a and De A aújo, 2021). Yang e al. (2013), obse ed a
signi ican dec ease o SAA (94 %) a a coppe concen a ion o 5 mg
L
−1
, esul ing in 0.022 g N g VSS
−1
d
−1
and cell lysis. Thei wo k
concluded ha he in luen coppe le el should no be highe han 4 mg
L
−1
o main ain a good pe o mance in long- e m con inuous- low sys-
ems (Yang e al., 2013). In a simila ange, 5 mg L
−1
o coppe nano-
pa icles educed anammox NRR by 85 % a e one mon h o incuba ion
in Zhang e al. (2018). Di e en esea che s de e mined median inhib-
i o concen a ions (IC
50
) in he ange o 5 mg Cu
2+
L
−1
in anammox
g anula sludge (Ak an e al., 2021; Li e al., 2015). Howe e , indus ial
was ewa e s can p esen a wide a ie y o Cu concen a ions: om
pha maceu ical e luen s, swine, o s eel manu ac u ing was ewa e s
wi h 0–33 mg Cu
2+
L
−1
o mine wa e s wi hin he ange o g Cu
2+
L
−1
(Li e al., 2018a, 2018b).
The p esen s udy aimed o in es iga e he use ulness o a combi-
na ion o sho ba ch es s wi h anammox g anules and en i onmen al
p o eomics o de ec ea ly-s age anammox s ess indica o s. We used as
an example o a po en ial s esso a hea y me al, coppe , in concen a-
ions be ween 0.1 and 5 mg L
−1
. This ea ly de ec ion wo k low can be o
g ea alue, especially o be implemen ed on acili ies as a ea abili y
es be o e dealing wi h new indus ial was ewa e .
2. Ma e ials and me hods
2.1. G anula sludge om anammox eac o
The anammox g anules we e collec ed om a pilo plan o 200 L
consis ing o a single uni , in he ELAN® p ocess de eloped by he
company FCC Aqualia in collabo a ion wi h he Uni e si y o San iago
de Compos ela. The in luen o he pilo consis s o ejec ed wa e om
he anae obic sludge diges e o he u ban WWTP loca ed in Guilla ei
(Galicia, Spain) con aining 540–1045 mg NH
4 +
-N L
−1
and ope a ed a
30 ◦C (Val del Río e al., 2017).
V. Guzm´
an-Fie o e al.
Science o he To al En i onmen 912 (2024) 169349
3
2.2. Ba ch es s
The es s we e ca ied ou in 105 mL se um ials con aining 63 mL
syn he ic was ewa e . Each assay u ilized a inal concen a ion o 0.5 g
VSS L
−1
o anammox g anula sludge. The mine al medium con ained:
KHCO
3
, KH
2
PO
4
, MgSO
4
H
2
O, CaCl
2
⋅2H
2
O, and ace elemen solu ions I
(EDTA 2 Na
2
H
2
O and FeSO
4
7H
2
O) and II (EDTA*2Na
2
H
2
O, ZnSO
4
7H
2
O, CoCl
2
7H
2
O, MnCl
2
4H
2
O, NaMoO
4
2H
2
O, NiCl
2
6H
2
O, Na
2
SeO
4
and H
3
BO
3
), as desc ibed by Van de G aa e al. (1996). The ini ial
concen a ions o NH
4
+
-N and NO
2
-N we e 100 mg L-1. The ace elemen
solu ions we e p epa ed wi hou coppe ha was hen added as CuSO
4
5H
2
O in concen a ions o : 0.1, 0.5, and 5 mg L-1 o Cu
2+
. Con ols
wi hou any coppe we e included. Fou eplica e mic ocosms we e se
up o each ea men (i.e.: coppe concen a ion).
The bo les we e hen placed in a he mos a ic shake a 150 pm.
The empe a u e was main ained a 32 ±1 ◦C and pH was adjus ed o
7.0 by adding HCl solu ion o he medium. Samples we e ob ained o e a
pe iod o 28 h using a sy inge needle o analyze he concen a ions o
NH
4
+
-N, NO
2
-N, and NO
3
-N. Ammonium (NH
4
+
), ni i e (NO
2
), pH, o al
suspended solids (TSS), and ola ile suspended solids (VSS) we e
quan i ied acco ding o he APHA S anda d Me hods (APHA, AWWA,
WEF, 2012). The NRR was calcula ed by conside ing he p oduc ion o
ni ogen gas based on he s oichiome y desc ibed by S ous e al.
(1998). This calcula ion in ol ed he consump ion o ammonia and ni-
i e as eac an s. Addi ionally, he SAA was calcula ed by no malizing
he ni ogen emo al a each ime poin wi h espec o he sludge
concen a ion in he assay (Dapena-Mo a e al., 2007). S a is ical ana-
lyses we e pe o med using analysis o a iance (ANOVA) wi h Tukey's
es as a pos hoc analysis wi h G aphPad P ism e sion 5.0 (G aphPad
So wa e, USA). P alues <0.05 we e conside ed s a is ically signi ican .
2.3. P o eome ex ac ion
A e each assay, 10 mL was collec ed a he inal incuba ion ime
(28 h) and cen i uged a 6000 pm o 10 min. Fi s , iplica e ac ions
o 150 mg o we pelle we e washed wi h PBS [137 mM NaCl, 2.7 mM
KCl, 10 mM Na
2
HPO
4
, 1.8 mM KH
2
PO
4
, pH 7.4] and hen esuspended
wi h ex ac ion bu e [50 mM T i's bu e , 1 % SDS (Aplichem Pan eac,
USA), pH 7.5]. Cell dis up ion was pe o med by sonica ion wi h an
ul asonic cell dis up o (B anson Soni ie S-150, USA) on ice a a
maximum o 40 % ene gy wi h an ou pu powe o 0.08 wa s RMS o
h ee cycles o 1 min o b eak agg ega es and lyse he cells. A e his, he
samples we e cen i uged (Eppendo cen i uge 5417R, Eppendo ,
Ge many) o 20 min a 3700 ×g a 4 ◦C. Finally, he supe na an was
collec ed. To concen a e he p o eins and o clean hem om bu e s,
sal s, o o ganic con aminan s a leas wo s eps o 10 % ichlo oace ic
acid (TCA, Sigma Ald ich) a 4 ◦C p ecipi a ion we e done. Then, he
ubes we e cen i uged (10 min, 9000 ×g, 4 ◦C) (Uni e sal 320, He ich,
Ge many), he supe na an was disca ded, and he ob ained pelle was
esuspended in 100 mM ie hylammonium bica bona e (TEAB) bu e ,
pH 8.5 (Sigma-Ald ich, Spain). P o ein quan i ica ion was done wi h he
Pie ce™ bicinchoninic acid assay (BCA) P o ein Assay Ki (The mo-
Fishe Scien i ic, USA), using a bo ine se um albumin (BSA) s anda d
cu e. T iplica e independen ex ac ions we e pe o med om each
mic ocosm and hen all samples belonging o each coppe ea men
we e pooled oge he be o e u he p o eomic analyses. The alues
p esen ed he e a e he a e age and he s anda d concen a ions o he
h ee indi idual ex ac s o each ea men be o e hei pooling.
2.4. SDS-PAGE elec opho esis
To check he simila i y o eplica es and he quali y o he samples (i.
e., p o ein deg ada ion), sodium dodecyl sul a e-polyac ylamide gel
elec opho esis in dena u ing condi ions (SDS-PAGE) was pe o med,
ollowing he p ocedu e de ailed in Kennes-Veiga e al., 2022. B ie ly,
elec opho esis was un o duplica e aliquo s wi h 10
μ
g p o ein/well.
P o ein samples we e mixed wi h NuPAGE LDS Sample Bu e and
NuPAGE Reducing Agen acco ding o manu ac u e ins uc ions. As a
molecula weigh ma ke , he PageRule ™ Plus P es ained Ladde om
10 o 250 kDa (The moFishe Scien i ic, USA) was included. Bis-T is
NuPAGE 4–12 % gel was used (The moFishe Scien i ic, USA) and
elec opho esis was un a 200 V. P o ein bands we e isualized using
he Impe ial™ P o ein S ain (The moFishe Scien i ic, USA) ollowing
he manu ac u e 's p o ocol.
2.5. P o ein analysis by mass spec ome y
A label- ee app oach was used o he quan i ica ion o he p o eins
p esen in each sample (Zhang e al., 2013). Fi s , samples we e ypsin-
diges ed, educed-alkyla ed, and inally desal ed using ZipTip-
μ
C18
ma e ial (Me ck, Ge many). The ob ained pep ide samples we e
analyzed by in-solu ion sho gun p o eomics (Zhang e al., 2013). Pep-
ide samples (0.2
μ
g o p o ein) we e injec ed on o a imsTOF P o mass
spec ome e (B uke , B emen, Ge many) equipped wi h a nano-
elec osp ay sou ce (Cap i eSp ay) and a ims-QTOF analyze . The
ch oma og aphic analysis was pe o med using a nanoELUTE ch o-
ma og aph (B uke ) wi h an Au o a analy ical column (C18, 250 ×
0.075 mm, 1.6
μ
m, 120 Å, IonOp icks). The nHPLC was con igu ed wi h
bina y mobile phases ha included sol en A (0.1 % o mic acid in miliQ
H
2
O) and sol en B (0.1 % o mic acid in ace oni ile). The analysis ime
was 105 min, in which he B/A sol en a io was g adually inc eased.
Blanks we e injec ed be ween samples wi h an analysis ime o 60 min,
con i ming no ca y-o e . Fo mass spec ome y (MS) acquisi ion, a
collision-induced dissocia ion (CID) agmen a ion and a nanoESI posi-
i e ioniza ion mode was employed. PASEF-MSMS scan mode was
es ablished o an acquisi ion ange o 100–1700 m/z (Qui on-Tapia
e al., 2023). Ma ches we e il e ed o 1 % alse disco e y a e (FDR) a
he pep ide le el. MS analyses we e pe o med a he Mass Spec ome y
and P o eomics Uni (A ea o In as uc u es) o he Uni e si y o San-
iago de Compos ela.
2.6. Me ap o eomic da a analysis
MS/MS spec a we e p ocessed wi h PEAKS S udio (Bioin o ma ics
Solu ions, Canada) so wa e o p o ein iden i ica ions and quan i ica-
ions based on he spec al coun ing me hod and he Spec alue. Due o
he na u e o hese samples (incomple ely cha ac e ized om a genomic
poin o iew) and he ocus o his s udy, we used a homemade da abase
(Zhang e al., 2013) wi h all p o ein sequences a ailable in NCBI p o ein
om he anammox gene a B ocadia, Je enia and Kuenenia (o de B o-
cadiales). The Compa e module om PEAKS S udio (so wa e PEAK
S udio 10.6, Bioin o ma ics Solu ions Inc., Canada) was used o p o ein
label- ee quan i ica ion using he sample Con ol as con ol. The alue
Spec p esen ed he e is he esul o his compa ison. MS da a was p o-
cessed in P o ein G oup mode. The mass spec ome y p o eomics da a
was deposi ed in he P o eomeXchange Conso ium ia he PRIDE
pa ne eposi o y wi h he da ase iden i ie PXD041756. As i s
s ingen il e ing, he i s p o ein o each p o ein g oup was manually
selec ed, and he o he s we e no conside ed. Also, only hose p o eins
iden i ied wi h a leas 2 unique pep ides we e conside ed in his s udy
(Zhao and Lin, 2010). G aphs we e made using G aphPad P ism 8.0
(G aphPad So wa e, USA). The alues on he hea maps we e no mal-
ized by he nex exp ession: Rowno m =
(Rowi−Mean)/(Rowmax −Rowmin). Whe e Row
i
ep esen s he ow o
he g oup con aining p o ein i o each coppe concen a ion. Row
min
and Row
max
deno e he minimum and maximum alues, espec i ely, o
he g oup o each coppe concen a ion (Galili e al., 2018). I Row
min
=Row
max
, Row
no m
=0. Fo he Venn diag am, he so wa e VENNY 2.1
was used (Oli e os, 2007-2015). GO ca ego iza ion and ee iew isu-
aliza ion o Ca. B ocadiaceae ela i e abundances was done in Unipep
Desk op .3.0. (Mesue e e al., 2015).
V. Guzm´
an-Fie o e al.
Science o he To al En i onmen 912 (2024) 169349
4
2.7. Basic local alignmen sea ch ool analysis (BLAST)
BLAST analysis was used a e he p o eomic pipeline o ind ho-
mologous o hose p o eins anno a ed as “hypo he ical p o eins”. The
BLAST ool a ailable a he NCBI websi e was used o compa e he lis ed
p o eins agains hose included in he da abase o “Candida us B oca-
diales” (NCBI axid:1127829). Signi ican simila i y was conside ed
when he ob ained E- alues we e below 1 ×10
−20
(Boleij e al., 2020).
3. Resul s and discussion
3.1. E ec o coppe concen a ions on anammox ac i i y
NRR o anammox g anules a di e en coppe concen a ions was
measu ed a e an incuba ion pe iod o 28 h (Fig. 1). >83 % o he ni-
ogen was consumed in all assays. The mean SAA o he con ol and he
es conduc ed wi h 5 mg L
−1
was 0.25 g N g VSS
−1
d
−1
, and no sig-
ni ican di e ence was obse ed be ween he SAA alues (p- alue =
0.5087, 95 % con idence in e al). I has been p e iously epo ed ha
in longe incuba ions simila coppe concen a ions led o dec eased
anammox ac i i ies. Fo ins ance, Yang e al. (2013) obse ed a signi -
ican dec ease in SAA (94 %), esul ing in 0.022 g N g VSS
−1
d
−1
and cell
lysis. Zhang e al., 2018 de ec ed a educ ion o anammox NRR 85 %
a e one mon h o incuba ion wi h coppe nanopa icles and Ak an e al.
(2021) de e mined an IC
50
o 6.7 mg L
−1
in long- e m (6.74 mg L
−1
)
expe imen s (240 days). We he e o e p edic ha a longe incuba ion
migh ha e led o educed anammox ac i i ies.
3.2. Anammox communi y composi ion
P o eome samples we e collec ed a e 28 h o incuba ions, ex ac-
ed, and analyzed by en i onmen al p o eomics. The p o ein concen-
a ion (
μ
g p o ein/
μ
L) de e mined by BCA on he iplica e p o eome
samples om each ea men we e 1.45 ±0.01 (0.1), 1.69 ±0.02 (0.5),
1.36 ±0.01 (5), 1.65 ±0.01 (0, Con ol), espec i ely. The esul s
p esen ed om he e on co espond o he combina ion o 3 indi idual
ex ac s. As quali y con ol o he p o eome samples, an SDS-PAGE
elec opho esis was pe o med con i ming he absence o p o ein
deg ada ion and a good ep esen a ion o p o eins om all he p o ein
sizes in all cases (Fig. S1). The numbe o p o eins iden i ied anges om
3996 (0.5 mg Cu L
−1
) o 5015 (con ol). The s uc u e o he anammox
popula ion was assessed in all he coppe concen a ions by calcula ing
he ela i e con ibu ion o pep ides belonging o each genus wi h
espec o he o al o pep ides iden i ied on each sample (Blakeley-Ruiz
and Kleine , 2022; Kennes-Veiga e al., 2022) (Fig. 2A). Ca. B ocadia
was he p edominan genus wi h a 92 ±0.2 % ela i e abundance, while
a mino p esence o Ca. Je enia (7.1 ±0.2 %) and Ca. Kuenenia (1.3 %
±0.1) was de ec ed in all cases. The e o e, he coppe concen a ions
applied in his s udy (0–5 mg Cu L
−1
) did no ha e an impac on he
abundance o any o he anammox gene a as expec ed conside ing he
ela i ely long doubling imes o anammox bac e ia (e.g., 7–11 days)
(Ka al e al., 2012) and he sho incuba ion ime applied in he p esen
s udy.
In all he cases, Ca. B ocadia ulgida was he p edominan species
om he genus (90.2 % ±0.7 %) (Fig. 2B). Ou p o eomic esul s ma ch
wi h p e ious s udies showing by me agenomic echniques ha Ca.
B ocadia ulgida is he p edominan mic oo ganism in anammox sludge
om he same WWTP used in his s udy, wi h ela i e abundances
anging om 80 % o 90 % (Mo ales e al., 2015).
3.3. P o eome iden i ica ions
A Venn diag am was c ea ed o display he numbe o common and
unique p o ein iden i ica ions ac oss di e en ea men s a e selec ing
only hose p o eins iden i ied wi h a leas 2 unique pep ides (Zhao and
Lin, 2010) (Fig. 3). The diag am e ealed ha 352 (53.3 %) p o eins
we e sha ed ac oss all samples. The samples wi h 0 (con ol) and 5 mg
L
−1
o coppe showed he highes numbe o unique p o eins in he
analysis, a 8.2 % and 7 %, espec i ely. In he Venn diag am, he unique
p o eins o each ea men a e ca ego ized acco ding o GO molecula
unc ion. The unique p o eins de ec ed on he 5 mg Cu L
−1
ea men
mainly belong o he ca ego y's me abolic pa hways and bac e ial
me abolism in di e se en i onmen s is mo e abundan . I is also wo h o
men ion he appea ance o p o eins om he wo-componen sys em.
3.4. Gene on ology (GO) p o ein ca ego ies had a di e en ial exp ession
in luenced by coppe concen a ion
Rega ding he subcellula loca ion o he p o eins de ec ed on he
me ap o eomic analyses, mos o hem belong o he memb ane ac ion
(42 %), ollowed by he cy oplasm (22 %), plasma memb ane (13 %),
ibosome (7 %) and anammoxosone (5 %) (Fig. 4). Anammox cell a -
chi ec u e is peculia as hese bac e ia ha bo a iple-laye ed mem-
b ane: pe iplasmic, cy oplasmic, and a specialized memb ane
en eloping he anammoxosome (De Almeida e al., 2016). This sophis-
ica ed a angemen and he co esponding memb ane-bound p o eins
Fig. 1. Ni ogen emo al a e o anammox g anules exposed o inc easing coppe concen a ions a e 28 h o exposu e.
V. Guzm´
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Science o he To al En i onmen 912 (2024) 169349
5
play a c ucial ole in he e iciency o hei espi a o y p ocesses. Among
ea men s, we de ec ed a di e en ial exp ession o memb ane ac ion,
cy oplasm, and ibosome mo e ep esen ed in he assays Con ol and
0.1 mg Cu L
−1
. On he o he hand, he exp ession o plasma memb ane
and ex acellula p o eins was inc eased in he highe coppe concen-
a ion (5 mg Cu L
−1
). The anammoxosome exhibi ed consis en
exp ession le els ac oss all coppe assays (Fig. 4). The anammoxosome
cons i u es he hea o he anammox ca abolism being so a , a unique
cell o ganelle ha alloca es he h ee main enzymes o ammonia and
ni i e anae obic oxida ion. The pe iplasm con ains he mos nume ous
and di e se coppe -dependen enzymes in bac e ia. Acco dingly, his
compa men is mos a isk o coppe -induced damage, which is exac-
e ba ed unde anoxic condi ions (Zhang e al., 2017).
Conside ing he molecula unc ion classi ica ion, me al ion binding
was he mos abundan ca ego y (17 %), ollowed by ATP binding (12
%), oxido educ ase ac i i y (8 %), heme binding (7 %), and elec on
ans e ac i i y (7 %) (Fig. S2). This is indica i e o he anammox
dependence on he me als p esen in he en i onmen and edox e-
ac ions. Me al ion binding p o eins a e s ongly associa ed wi h elec on
ans e o edox ca alysis, essen ial in anammox ene gy me abolism
(Fe ousi e al., 2019). Ex acellula p o eins om anammox g anules
ha e been obse ed o exhibi a as e esponse in binding Cu
2+
han
polysaccha ides and hyd oca bons, wi h ca boxyl g oups playing a sig-
ni ican ole. (Li e al., 2020). A ecen s udy in es iga ed he mul ime al
adso p ion capaci y o EPS p o ein om anammox g anula sludge and
ound ha he adso p ion capaci y o coppe was highly dependen on
he ex acellula ma ix (Pagliaccia e al., 2022).
Fig. 2. A: Rela i e abundance o each anammox gene a in he p o eomes om each Cu ea men . B: T ee iew isualiza ion (Mesue e e al., 2015) o he ela i e
abundance o Ca. B ccadia membe s a 5 mg Cu
2+
/L.
Fig. 3. Venn diag am showing he numbe o p o eins iden i ied in Anammox sludge a e incuba ion wi h di e en coppe concen a ions. The in e sec ion o
ellipses indica es he numbe o p o eins sha ed be ween samples.
V. Guzm´
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Science o he To al En i onmen 912 (2024) 169349
6
3.5. Ni ogen me abolism
Those p o eins known o be in ol ed in anammox ni ogen me a-
bolism we e speci ically sea ched o in his s udy. The esul s a e
de ailed he e ollowing he same ack as a ni i e and ammonium
molecule would ollow. Fi s , he ni i e anspo e (FocA) and he
ammonium anspo e (Am B) a e localized on he anammoxosome
memb ane and a e esponsible o impo ing he ni ogen species in o
he co e anammox ene gy machine y. This dis inc i e cellula s uc u e,
e med he anammoxosome, con ains h ee enzymes ac ing in he
ollowing o de : i) ni i e educ ase (Ni ), which con e s ni i e in o
ni ic oxide (NO), ii) hyd azine syn hase (Hzs), which combines NO and
ammonium o o m he singula in e media e compound hyd azine
(N
2
H
4
) and iii) hyd azine dehyd ogenase (Hdh), o ming dini ogen gas
om hyd azine (Ka al and Kel jens, 2016).
In all he p o eomes analyzed, he ni i e anspo e FocA
(KKO18363.1) and he ni i e educ ases om B. ulgida we e de ec ed
among he 4–6 mos abundan p o eins in all samples (Table 1). The
ammonium anspo p o ein (Am B, RIK00381.1), localized on he
anammoxosome memb ane was also de ec ed in all samples (al hough
in di e en anking posi ions).
Hyd azine syn hase (Hzs) is esponsible o he so-called anammox
eac ion: he con e sion o ammonium and ni ic oxide o hyd oxyl-
amine o p oduce he oxic hyd azine. Hzs is a unique enzyme om
anammox, conside ed a biochemical no el y, and i s unc ion canno be
eplaced by o he p o eins, making his gene an app op ia e unc ional
bioma ke o he de ec ion o hese bac e ia by PCR (Yang e al., 2018).
This enzyme is an he e o ime , encoded by he hzsCBA gene clus e ,
and in ou p o eome samples, HzsA (AEW50030.1) and HzsC
(KKO20885.1) om Ca. B ocadia ulgida we e always among he h ee
mos abundan p o eins (Tables S1-S4), indica ing he ex eme deg ee o
specializa ion o anammox cells on his ene gy me abolism.
Hyd azine dehyd ogenases (Hdh) (KKO18553.1) we e exp essed in
all samples. This homo ime ic enzyme ca alyzes hyd azine oxida ion
and he e o e p oduces abou hal o all N
2
emi ed in o he a mosphe e
(Ka al e al., 2012). Hdh a e i on-dependen and belong o he HAO-like
oc aheme p o eins being he e o e ela ed o he hyd oxylamine
oxido educ ase (HAO) om ae obic ammonium-oxidizing bac e ia (De
Almeida e al., 2016; Ka al e al., 2011) and in he public genome o Ca.
B ocadia ulgida hey a e anno a ed as HAO. The exp ession o he in-
di idual p o eins om he anammoxosome was no a ec ed by he
coppe concen a ion, as poin ed ou in he GO analysis de ailed in
Sec ion 3.4. This is di e en o wha desc ibed in he case o empe a u e
s ess in Wang e al., 2021, whe e Hzs was signi ican ly less exp essed
a e a empe a u e d op om 35 ◦C o ◦15 ◦C. Also, he p esence o high
concen a ions o o ganic compounds such as p-ni ophenols has been
shown o dec ease he exp ession o exp ession o Hzs and o he
Fig. 4. Hea map ep esen ing he Gene On ology (GO) classi ica ion o he p o eins de ec ed in he anammox sludge a e 28 h in he p esence o he di e en coppe
concen a ions applied, acco ding o he ca ego ies o Cellula componen s. The co esponding numbe o o al pep ides iden i ied o each ca ego y accompanies
each hea map on he igh Y axis.
Table 1
Anammox p o eins in ol ed in he ni ogen me abolism de ec ed in he p esen
s udy by me ap o eomics and hei Spec alue a he end o he expe imen s in
he di e en coppe ea men s. The Spec alue is based on pep ide spec um
ma ches (PSM) and was used as an indica o o he ela i e abundance o he
p o eins in each sample.
Ni ogen me abolism
P o ein Accession Con ol 0.1 0.5 5
Ni i e anspo e - FocA KKO18363.1 8 4 0 7
Ammonium anspo e – Am B RIK00381.1 19 13 9 10
Ni i e educ ase - Ni OQZ00558.1 206 215 196 207
Ni i e educ ase - Ni KKO18751.1 186 150 108 123
Hyd azine syn hase - HzsA AEW50030.1 679 557 592 703
Hyd azine syn hase - HzsB BBO18371.1 35 29 37 32
Hyd azine syn hase - HzsC KKO20885.1 243 234 240 286
Hyd azine dehyd ogenases -
Hdh
KKO18553.1 116 100 83 100
Heme anspo e CcmC OQZ02497.1 291 261 254 299
Pe iplasmic ni a e (o ni i e)
educ ase - NapC_ni T
UJS19330.1 32 30 27 22
V. Guzm´
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Science o he To al En i onmen 912 (2024) 169349
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anammoxosome enzymes in Luo e al., 2022.
The heme anspo e CcmC (OQZ02497.1) was also among he h ee
mos abundan p o eins in all p o eome samples. Heme c is a e y
impo an co- ac o in he main anammox me abolic eac ions wi h
ca aly ic and elec on- ans e po en ial and is esponsible o he
cha ac e is ic ca min colo on en iched anammox biomass. Ma e al.
(2019) showed how heme C concen a ion measu ed by a
spec opho ome y-dedica ed me hod was posi i ely co ela ed wi h
NRR in anammox cul u es and he e o e also a possible indica o o
e alua e anammox pe o mance. In ou s udy, he abundance o his
i on-dependen cy och ome did no show a end ela ed o he p esence
o coppe (Table 2) poin ing o a gene al good i ness on he anammox
ene gy sys em. These esul s also demons a e ha he use o me a-
p o eomics o de ec anammox-speci ic ac i i ies in enginee ed sys ems
is echnically possible.
In e es ingly, we also de ec ed a p o ein anno a ed as ‘napC_ni T
pe iplasmic ni a e (o ni i e) educ ase c- ype cy och ome’ (UJS19330.1).
Acco ding o he NCBI, nea ly e e y membe o his sub amily is a ni-
a e educ ase, speci ically NapC. This is a p edic ed memb ane-
ancho ed ou -heme c- ype cy och ome ha o ms one componen o
he pe iplasmic ni a e educ ase along wi h o he NapABDEF subuni s.
A single known excep ion cu en ly is Ni T, which is a componen o a
ni i e educ ase. To elucida e he implica ion o his exp essed enzyme
on ni i e o ni a e educ ion would equi e u he esea ch e o .
3.6. Anammox cen al ca bon me abolism
The cen al ca bon me abolism o anammox was esol ed by Lawson
e al. (2021) using ca bon and hyd ogen iso ope acing and me ab-
olomics. Anammox a e no able o use ace a e as ca bon o ene gy sou ce
in si u and hese bac e ia make use o he Wood Ljungdahl/ace yl-
coenzyme A (CoA) pa hway o ca bon ixa ion. In his pa hway, high-
ene gy elec ons om hyd azine a e ans e ed ia e edoxin o he
ace yl-CoA syn he ase/CO dehyd ogenase, and he eplenishmen o he
hyd azine pool o compensa e he in es men in ca bon ixa ion is
achie ed by e e se elec on anspo . The ac i i y o he oxida i e
b anch o he ica boxylic acid cycle o alpha-ke oglu a a e biosyn-
hesis and he di ec assimila ion o ex acellula o ma e ia he Wood-
Ljungdahl pa hway ins ead o i s comple e oxida ion o CO
2
ollowed by
eassimila ion was con i med.
E en hough no ca bon sou ce was added o he medium in he
p esen s udy we de ec ed he ollowing enzymes om he Wood
Ljungdahl pa hway: CO dehyd ogenase/ace yl-CoA syn hase
(KKO18855.1), alcohol dehyd ogenase (RIJ91218.1), me hyl ans e ase
(RIJ92330.1), me hylene-H4F-dehyd ogenase (OQY97878.1), o myl-
H4F syn hase (KKO19470.1) and o ma e dehyd ogenase
(OQY98926.1). No enzyme om he TCA cycle was ound exp essed and
om he gluconeogenesis, he phosphoglyce a e kinase (UJS20358.1),
uc ose-1,6-bisphospha ase (UJS19600.1), and glucose-6-phospha ase.
3.7. S ess esponse
Despi e he homogeneous NRR ound in he di e en coppe ea -
men s and he s able exp ession o he anammoxosome, we de ec ed an
inc ease in he exp ession o speci ic p o eins p e iously linked o s ess
condi ions. The abundance o he p o ein anno a ed as ‘Mul icoppe ox-
idase domain-con aining p o ein’ om Ca. B ocadia sp. (UJS22045.1.)
inc eased in a manne di ec ly p opo ional o he coppe concen a ion
(Fig. 5). The link be ween his p o ein and he gene al mechanism o
p o ec cells agains he oxici y o he coppe accumula ed in he
pe iplasm has been p e iously well-desc ibed in clinical bac e ia such as
E. coli o Mycobac e ium ube culosis (Ladome sky and Pe is, 2015).
O he s ess- ela ed p o eins, such as bac e io e i in, uni e sal s ess
p o ein, o supe oxide dismu ase showed he highes exp ession on he
highes coppe concen a ion (5 mg L
−1
), ye hey did no ollow such a
clea end as he wo p e iously discussed (Table 2). Addi ional p o-
eins ela ed o s ess in anammox cells in Zo z e al. (2018), i.e., speci ic
chape ones, anspo e s, ATPases, alkyl hyd ope oxide educ ase,
ube yh in amily-p o ein and B12-binding domain-con aining adical
SAM p o ein we e p esen in ou samples, ye no di e en ially
exp essed in ega ds o he coppe ea men (Table S1-S4).
Unexpec edly, we also de ec ed an inc eased exp ession o he ‘RNA-
me abolising me allo-be a-lac amase (MBL)’ (KKO20060.1) in esponse
o coppe (Fig. 5). We did no add any β-lac ams in ou mic ocosms and,
e en when anammox g anules we e collec ed om a WWTP ea ing
u ban was ewa e , hey we e washed be o e p o ein ex ac ion. I is,
he e o e, imp obable he p esence o an ibio ics in he expe imen s and
hei ac i i y migh be di e en han β-lac amase. Blas ing o i s ull
aminoacidic sequence in NCBI gi es he highes iden i ies wi h o he
‘MBL old me allo-hyd olases’ bo h om he genus Ca. B ocadia. In e -
es ingly, hese enzymes we e ecen ly ound o ha e p omiscuous ac-
i i ies such as hyd olyzing a wide ange o subs a es, like DNA o RNA,
apa om β-lac ams and ha e been ound in humans, a chaea, ungi,
and now i uses (Colson e al., 2020). This is di e en om he d as i-
cally simpli ied pa adigm o enzymes wi h β-lac amase ac i i y being
exp essed by bac e ia de i ed om he selec i e p essu e o na u al o
p esc ibed an ibio ics (Colson e al., 2020).
The eason why i s exp ession was ela ed o he coppe concen a-
ion is also no e iden o us. MBL lac amases a e dependen on pe i-
plasmic zinc ions o ca alyze he hyd olysis o β-lac ams. Zinc ions can be
exchanged wi h cadmium, cobal , and manganese main aining ca aly -
ically ac i e enzymes (Page and Bada au, 2008). Howe e , Djoko e al.
(2018) showed ha coppe ions inhibi he ac i i y o MBL in i o and
enhance β-lac ams suscep ibili y o E. coli isola es ha bo ing MBL. I
would he e o e dese e mo e esea ch e o o elucida e he anammox
MBL ac i i y and i s in e ac ion wi h coppe in he condi ions used in
his s udy.
3.8. Speci ic p o eins esponsible o EPS p oduc ion inc eased in he
p esence o coppe
The p o eins ‘algina e expo amily p o ein’ (AlgE) and ‘poly-
saccha ide biosyn hesis p o ein’ we e de ec ed only in he genus B o-
cadia and in e es ingly, hei exp ession was augmen ed in hose
mic ocosms wi h supplemen ed coppe (Table 2). Algina e is an exo-
polysaccha ide known o be pa o he suga -based pa o he EPS o
anammox. Addi ionally, he p o ein anno a ed as ‘sul o ans e ase’
(accession UJS22315.1) om Ca. B ocadia and linked o he syn hesis o
sul a ed glycosaminoglycans (o glycop o ein) in anammox EPS (Mo e
Table 2
Selec ed anammox p o eins ela ed o s ess and EPS p oduc ion iden i ied by
sho gun me ap o eomics in his s udy ha showed an inc eased exp ession in
esponse o he inc easing coppe concen a ions. The Spec alue o each
p o ein on each coppe ea men concen a ion (mg L
−1
) is shown. The colo s
a e indica i e o he abundance, om ed (less abundan ) o g een (mos
abundan ).
S ess- ela ed p o eins
P o ein
Accession
Con ol
0.1
0.5
5
Uni e sal s ess p o ein
KKO18817.1
21
20
18
26
Bac e io e i in
RIJ99420.1
11
19
16
25
Supe oxide dismu ase
OQY97949.1
47
36
36
54
Mul icoppe oxidase domain-con aining
p o ein
UJS22045.1
0
4
9
15
RNA-me abolising me allo-be a-
lac amase
KKO20060.1
8
10
17
23
EPS P oduc ion
P o ein
Accession
Con ol
0.1
0.5
5
Algina e expo amily p o ein - AlgE
KKO20581.1
212
211
253
331
Algina e expo amily p o ein - AlgE
WP_052562998.1
79
104
115
114
Polysaccha ide biosyn hesis p o ein
UJS22320.1
0
0
3
6
Sul o ans e ase
UJS22315.1
0
0
0
3
V. Guzm´
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Science o he To al En i onmen 912 (2024) 169349
8
e al., 2014) was de ec ed only a he highes coppe concen a ion
applied (5 mg L
−1
) wi h a Spec alue o 3 (Table 2). Ou esul s indica e
a highe p oduc ion o EPS om Ca. B ocadia in esponse o coppe , as i
was p e iously shown in o he bac e ial gene a such as En e obac e
ae ogenes o Pseudomonas pu ida (Mo e e al., 2014) and o anammox
g anules in se e al s udies (Zhang e al., 2015). Zhang e al. (2015)
in es iga ed he ole ance mechanism o g anula anammox bac e ia
agains coppe by ex ac ing he EPS and quan i ying i s o al p o ein
con en by he Low y me hod and ound ha highe coppe concen a-
ions igge an inc ease in p o ein le els wi hin he EPS, e ec i ely
shielding he anammox cells. In ou s udy, we de ec ed an inc ease in
p o eins loca ed in he ex acellula ma ix and addi ionally, we ha e
iden i ied which speci ic p o eins a e inc easing as an ea ly-s age
esponse o he p esence o s essing concen a ions o coppe . Apa
om his unde lying mechanism o coppe sel -p o ec ion, i was p o-
posed ha anammox cells sel -heal by ac i ely pumping hea y me als
ou o he mos sensi i e egions o he cells, i.e., ac i e e lux mecha-
nisms (Zhang e al., 2016). We did no de ec an inc ease in any o he
e lux pumps o memb ane anspo e s. The e o e, he esul s o ou
s udy poin o he i s mechanism as an ea ly esponse o he p esence o
inhibi o y coppe concen a ions in anammox g anules.
An inc ease in anammox EPS p oduc ion has also been demons a ed
in esponse o o he me als, such as nickel o cadmium (Zhang e al.,
2023). Bo h a e o g ea ele ance o indus ial acili ies, as nickel is
widely used in elec opla ing, ca alys s, pain s, and o he ields, while
cadmium is p esen in me al e ine y discha ges, was e ba e ies uno
o pes icides manu ac u ing wa e s among o he s. Simila ly, salini y has
been shown o inc ease he EPS p oduc ion and al e he communi y
s uc u e o anammox sys ems in Wu e al., 2019. In he p esen con ex
o a clima ic c isis, salini y is a e y ele an pa ame e o be conside ed.
O he physical-chemical pa ame e s ha ha e been desc ibed o al e
he mic obial ac i i ies, and mo e speci ically he p o eome exp essions
in anammox a e empe a u e o pH (Izadi e al., 2021). Also, o ganic
compounds such as ni ophenols ha e been desc ibed o educe he EPS
con en on anammox g anules, al e ing he se ling p ope ies o hei
g anules (Luo e al., 2022).
4. Conclusions
The inc easing concen a ions o he con aminan applied o he
anammox g anules led o in e es ing indings a he p o eome le el.
Fi s , in ag eemen wi h he mac oscopic esul s (simila NRR), anam-
moxosome enzymes exp essions we e s able in all coppe ea men s.
The hyd azine syn hase is widely conside ed a molecula bioma ke o
anammox ac i i y and was he mos abundan enzyme in all p o eomes.
This ac p o es ha en i onmen al p o eomics can be applied o
acking his mic obial ac i i y in enginee ed sys ems. This is o in e es
as p e ious s udies ha e shown impac s on he hyd azine syn hase ac-
i i y by empe a u e d ops o he p esence o o ganics such as ni o-
phenol. Fo he ea ly de ec ion o coppe -de i ed s ess, o he p o eins
we e ound o be mo e use ul. Speci ic anammox p o eins ela ed o
s ess and EPS p oduc ion inc eased hei abundance p opo ionally o
coppe concen a ion. Mo eo e , ano he g oup o s ess- ela ed p o-
eins showed hei highes exp ession a 5 mg Cu L
−1
. The de ec ed in-
dica ions o anammox s ess a e in ag eemen wi h p e ious li e a u e
epo s o coppe IC
50
alues in he ange o 5 mg Cu L
−1
. Ou esul s
sugges ha en i onmen al p o eomics is echnically use ul o de ec ing
ea ly-s age s ess bioma ke s in anammox bac e ia. This me hodology
appea s o p o ide mo e in o ma ion ega ding pollu an s' e ec s on
bac e ia han a ge ed echniques, such as qPCR, ha only de ec spe-
ci ically selec ed genes (e.g.: hyd azine syn hase). The nega i e e ec o
coppe on anammox p o eomes was de ec able a e an exposu e ime as
sho as 28 h. and his sugges s ha he me hodology desc ibed in his
s udy can, he e o e, be ansla ed in o ele an ime and consequen ly
economic sa ings in was ewa e ea men acili ies leading o indus ial
was ewa e . The p oposed app oach would consis o es ing he new
e luen s o be ea ed wi h his me hodology be o e ull-scale bio-
eac o s a e se up o loaded. The me hodology applied he e o he
s udy o coppe e ec s can be ans e ed o he s udy o he e ec o
o he con aminan s such as di e en hea y me als (including cadmium,
nickel, and o he s), o ganics such as ni ophenol, o he ola ile a y
acids p esen in high concen a ions on he e luen o anae obic
diges o s o en subjec ed o anammox ea men s. Also, changes in
physical-chemical pa ame e s such as empe a u e, pH, o salini y a e
expec ed o lea e a p o eomic ace, acco ding o p e ious li e a u e
epo s. This s udy also opens he doo o explo ing his me hodology on
o he g oups o bac e ia ele an o was ewa e ea men (e.g.: AOB,
ammonia-oxidizing bac e ia) o o he bio echnological p ocesses.
CRediT au ho ship con ibu ion s a emen
Víc o Guzm´
an-Fie o: Fo mal analysis, In es iga ion, W i ing –
o iginal d a , W i ing – e iew & edi ing. Albe o Dieguez-Seoane:
In es iga ion, W i ing – e iew & edi ing. Ma lene Roeckel: Concep-
ualiza ion, In es iga ion, P ojec adminis a ion, Resou ces, Supe i-
sion, W i ing – e iew & edi ing. Juan M. Lema: Concep ualiza ion,
Fo mal analysis, P ojec adminis a ion, Supe ision, Valida ion,
W i ing – e iew & edi ing. Alba T ueba-San iso: Concep ualiza ion,
Fig. 5. Impac o coppe concen a ion on he spec al pep ide ma ch coun s o he ‘mul icoppe oxidase domain-con aining p o ein’ (UJS22045.1) and he ‘RNA-
me abolising me allo-be a-lac amase’ (KKO20060.1) om anammox g anules.
V. Guzm´
an-Fie o e al.
Science o he To al En i onmen 912 (2024) 169349
9
Da a cu a ion, Fo mal analysis, In es iga ion, Me hodology, P ojec
adminis a ion, Supe ision, Valida ion, Visualiza ion, W i ing – o ig-
inal d a , W i ing – e iew & edi ing.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgmen s
In memo y o An onio T ueba de la Iglesia (Tio Comino). The au ho s
hank FCC Aqualia o he pa ial ni i a ion-anammox ELAN® g anula
biomass samples p o ided ha allowed hem o pe o m his s udy.
Ma lene Roeckel and Víc o Guzm´
an-Fie o we e suppo ed by Agencia
Nacional de In es igaci´
on y Desa ollo [1200583 (FONDECYT) and
2018-21180541]. Juan M. Lema Rodicio and Alba T ueba-San iso
belong o he Galician Compe i i e Resea ch G oup (GRC) ED431C-
2021/37. Alba T ueba-San iso acknowledges a Juan de la Cie a-Fo -
maci´
on pos doc o al G an (FJC2019-041664-I).
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.sci o en .2023.169349.
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