Hydroxylamine metabolism of Ca. Kuenenia stuttgartiensis

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Hydroxylamine metabolism of Ca. Kuenenia stuttgartiensis

Soler-Jofra, Aina; Laureni, Michele; Warmerdam, Marieke; Pérez, Julio; van Loosdrecht, Mark C.M.

DOI

10.1016/j.watres.2020.116188

Publication date

2020

Document Version

Final published version

Published in

Water Research

Citation (APA)

Soler-Jofra, A., Laureni, M., Warmerdam, M., Pérez, J., & van Loosdrecht, M. C. M. (2020). Hydroxylamine

metabolism of Ca. Kuenenia stuttgartiensis. Water Research, 184, [116188].

https://doi.org/10.1016/j.watres.2020.116188

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ContentslistsavailableatScienceDirect

Water

Research

journalhomepage:www.elsevier.com/locate/watres

Hydroxylamine

metabolism

of

Ca.

Kuenenia

stuttgartiensis

Aina

Soler-Jofra

a,∗

_,

_Michele

_Laureni

a

_,

_Marieke

_Warmerdam

a

_,

_Julio

_Pérez

b

_,

_Mark

_C.M.

_van

Loosdrecht

a

a Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Van der Maasweg 9, Delft 2629 HZ, the Netherlands b Department of Chemical, Biological and Environmental Engineering, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain

a

r

t

i

c

l

e

i

n

f

o

Article history: Received 2 May 2020 Revised 10 July 2020 Accepted 14 July 2020 Available online 15 July 2020 Keywords: Anammox Intermediate Disproportionation Hydrazine Nitrate

a

b

s

t

r

a

c

t

Hydroxylamineisakeyintermediateinseveralbiologicalreactionsoftheglobalnitrogencycle.However, theroleofhydroxylamineinanammoxisstillnotfullyunderstood.Inthiswork,theimpactof hydroxy-lamine(alsoincombinationwithothersubstrates)onthemetabolismofaplanktonicenrichmentculture oftheanammoxspeciesCa.Kueneniastuttgartiensiswasstudied.Anammoxbacteriawereobservedto produceammoniumbothfromhydroxylamineandhydrazine,andhydroxylaminewasconsumed simulta-neouslywithnitrite.Hydrazineaccumulation-signatureforthepresenceofanammoxbacteria-strongly dependedontheavailablesubstrates,beinghigherwithammoniumandlowerwithnitrite.Furthermore, the resultspresented here indicatethat hydrazine accumulation isnot the result ofthe inhibitionof hydrazinedehydrogenase,ascommonlyassumed,buttheproductofhydroxylaminedisproportionation. Allkineticparametersfortheidentiﬁedreactionswereestimatedbymathematicalmodelling.Moreover, thesimultaneousconsumptionandgrowthonammonium,nitriteandhydroxylamineofanammox bac-teriawasdemonstrated,thiswasaccompaniedbyareductioninthenitrateproduction.Ultimately,this study advancesthe fundamental understanding ofthemetabolic versatilityofanammox bacteria,and highlightsthepotentialroleplayedbymetabolicintermediates(i.e.hydroxylamine,hydrazine)inshaping naturalandengineeredmicrobialcommunities.

1. Introduction

Anaerobic ammoniumoxidizingbacteria(anammox)were first reportedinthe90sinawastewatertreatmentplant(Mulderetal., 1995). Anammox bacteria autotrophically oxidize ammonium to dinitrogengaswithnitriteaselectron-acceptor(Jettenetal.,1998). Before their discovery, even ifpredicted thermodynamically, am-monium activation in absence of oxygen had never been iden-tified in nature (Broda, 1977). Since then, significant efforts f o-cusedonunderstandingthecentral metabolismofanammox bac-teria(e.g.(Kartaletal.,2011;Oshikietal.,2016;Strousetal.,1998;

VanDeGraafetal.,1997)).

Initially, hydroxylamine was hypothesized to be an obligate intermediate of anammox catabolism, and hydrazine wasshown to accumulate when hydroxylamine wasadded inanammox cul-tures (Van De Graaf et al., 1997). More recently, NO was pro-posed to be the actual intermediate in the catabolic pathway (Kartaletal.,2011).Thecurrentworkinghypothesisforthe

anam-∗ _{Corresponding author.}

E-mail address: a.solerjofra@tudelft.nl (A. Soler-Jofra).

moxmetabolisminvolvesthreereactions.First,nitriteisconverted to NO (Eq.(1)) via a nitric reductase (Nir) enzyme. Then, NO is usedtoactivateNH4+andformtheN-Nbondneededtoproduce

hydrazine(N2H4)(Eq.(2))catalysedby hydrazinesynthase(HZS).

Finallyhydrazineisfurtherconvertedtodinitrogengas(Eq.(3))by hydrazinedehydrogenase(HDH).

NO−₂+2H++e− →NO+H2O (1)

NO+ NH₄++2H++3e− →N2H4+H2O (2)

N2H4 →N2+4H++4e− (3)

However, even with NO as the central intermediate, hydrox-ylamine seems to play a key, yet elusive role in anammox metabolism.Forexample, Ca. Brocadiaspp.strainsdo notencode for a Nir enzyme (Oshiki et al., 2015). Thus, either Ca. Brocadia spp. have hydroxylamine ascentral intermediate as proposed by Oshikiandcoworkers(Oshikietal.,2016),oranotherenzyme(like kustc0458)ratherthan Nir is convertingnitrite to NO (Hu etal., 2019). Moreover, different studies showed that one of the most highly expressed enzyme in anammox bacteria is the

hydroxy-https://doi.org/10.1016/j.watres.2020.116188

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lamineoxidase(HOX;kustc1601),whichisproposedtoconvert hy-droxylaminetoNO(Huetal.,2019;Kartaletal.,2011).Thereason foranammoxbacteriatoinvestenergytoexpressHOXatsuchhigh levelshasbeenhypothesizedtobethepossibilitytoreuseany hy-droxylamineleakingfromHZS(Dietl etal.,2015;Kartaland Kelt-jens,2016).Brieﬂy,theactual reactionmechanismsofHZSis pro-posedtoinvolveNOtransformationtohydroxylamine,and ammo-niumandhydroxylamine reactingto formhydrazine(Dietl etal., 2015;KartalandKeltjens,2016).Overall,theroleofhydroxylamine inanammoxmetabolismremainspoorlyunderstood.

In the environment where anammox bacteria thrive, free hy-droxylaminehasbeenmeasured((Huetal.,2017;Liuetal.,2017;

Poot et al., 2016; Soler-Jofra et al., 2018; Stüven et al., 1992;

Suetal.,2019;Teradaetal.,2017;YangandAlleman,1992;Yuand Chandran,2010;Yuetal.,2010;Yuetal.,2018)).Forinstance, hy-droxylaminehasbeenshowntotransientlyaccumulatein concen-trationsrangingfrom0.006-1mg-N/Lindifferentammonium oxi-dizingbacteria(AOB)purecultures (Liuetal.,2017;Stüvenetal., 1992; Yu andChandran,2010;Yu etal., 2010;Yu etal., 2018) or mixedconsortia(Huetal.,2017;Pootetal.,2016;Soler-Jofraetal., 2018;Suetal.,2019;Teradaetal.,2017;YangandAlleman,1992). Thus, anammox exposure to external hydroxylamine cannot be ruled out, inparticular in bioﬁlm systems, where hydroxylamine canbe produced by anitrifying populationandcan reach higher concentrationsthaninthebulkliquid(Sabbaetal.,2015).Inthese systems,hydroxylaminecanbe producedintheexternaloxic lay-erswhereAOBare present,diffusethroughthebioﬁlm andreach theanoxic(anammoxbacteria)layers(Sabbaetal.,2015).

Batch testswithhydroxylamineadditionare generallyusedto studytheshorttermeffectsofhydroxylamineonanammox bacte-riaprimarilyto(i)demonstrateanammoxactivity(Eglietal.,2001;

Jettenetal.,1998),and(ii)studythe“boosting” effectof interme-diatesonanammox activity(Huetal., 2011; Zekker etal., 2012). However,tothebestofourknowledge,theeffectofdifferent sub-stratecombinationsonanammoxhydroxylamineconsumptionhas notbeendedicatedly studied.The onlyindepth studyonthe ef-fectof hydroxylamineonanammox metabolism usedammonium assoleco-substrate(vanderStaretal.,2008b).

In the present work, the impact of different combinations of substrates together with hydroxylamine on a planktonic anam-mox Ca. Kuenenia stuttgartiensisculture inbatch testswas stud-ied.Also, thelong-term impacts of hydroxylamineaddition were studied,andtheeffects on stoichiometryandmicrobial composi-tionwerequantiﬁed.Finally,athermodynamic andmodelling ap-proachwasdevelopedtoestimatethekeykineticparameters,and furtherunderstandtheanammoxmetabolismwithhydroxylamine as(co-)substrate.

2. Materialsandmethods

2.1. Batch test, preparation and procedure

Biomass was collected from a 10L MBR highly enriched in planktonic Ca. Kuenenia stuttgartiensis (79±4 % as estimated by 16SrRNA gene-basedamplicon sequencing analysis) (see Supple-mentaryInformation(SI))andcentrifugedfor15minutesat4200 rpmatroom temperature.Cellswerere-suspended inN2-sparged

mineralmediumtothedesiredbiomassconcentration.Themineral mediumhadthesamecompositionasdescribedinSI,butwithout ammonium or nitrite and supplemented with 1g/L NaHCO3. The

pHand optical density at660nm (OD660) were measured before

aliquoting50mLofcell suspension among 112mL serumbottles. Theoptical densitywascorrelated withgVSS/L(Fig. S1). Biomass usedinnegativecontrolswasboiledfor5minutesbefore aliquot-ing.Thebottlesweresealedwithrubberstoppers,andanoxic con-ditionswere achievedby sparging (ca. 1 minute)and vacuuming

(ca. 3 minutes) with Argon three times per bottle. Bottles were placedonashaker(Incubator HoodTH30,EdmundBühlerGmbH, Bodelshausen, Germany) at 30°C and 170 rpm. The pH was not adjusted, but remainedbetween 8.0 and8.5, within the optimal rangeforanammoxbacteria(Kartaletal.,2012).Bottleswere incu-batedovernight withstoichiometric concentrationsofammonium andnitritetoensureactivity.

After overnight incubation, the experiment wasperformed by adding a pulse ofammonium, nitrite, hydroxylamine, and/or hy-drazine from anoxic stock solutions to reach the desired initial concentrationineachbatchtest(seeTable1).Samplingwasdone overtimedependingonthebiomassactivitybyremoving4mLof cell suspension witha syringe. Sampleswere centrifuged for3-5 minutes (4200 rpm, 4°C), andthe supernatantwas kept for fur-ther analysisofthe dissolved nitrogen compounds (see SI). Sam-ples used for hydrazine (Watt and Chrisp, 1952) and hydroxy-lamine(FrearandBurrell, 1955)determination were treatedwith 200 μL of a 0.1 g/mL sulfamic acid solution to remove the dis-solvednitrite.Nitritewaspreviouslyshowntointerfereinthe hy-droxylaminedetermination(Soler-Jofraetal.,2016).Inthepresent study,nitritewasalsoshowntointerferethehydrazine measure-ment(seeFig.S2).Nitrogenconsumption/productionrates calcula-tionsaredescribedinSI.Microbialcommunitydynamicswerenot followed duringthebatch tests, butthey wouldnot be expected toshiftsigniﬁcantlyasnosigniﬁcant changeswereobserved dur-inglongtermhydroxylaminefeedinginacontinuousreactor(see

Section3.2).

2.2. Batch tests, thermodynamic analysis

Basedonthebatch testdata,thethermodynamicsofthe puta-tive reactionsinvolved inhydroxylamineconsumptionwere stud-ied(seeTable2).Reactions3,6,7,10and11were originally pos-tulatedby vanderStar(vanderStaretal.,2008b).Halfreactions were derivedbasedonN,O, Handcharge balances (seeexample inSI,basedon(KleerebezemandVanLoosdrecht,2010)).The stan-dardGibbsenergychange(

Go

R)ofeachreactioninTable2was

calculated with the standard Gibbs energy of formation (G°f) of

eachcompound(TableS1 andEq.(S1)).

Go

Rwascorrectedwith

themeasured concentrationofeach compoundateachbatch test time point to obtain the actual Gibbs energy change of reaction (

G1

R) evolution duringthe batch tests (Eq. (S2) and(S3)). The

aimwastodetectanypossiblethermodynamicslimitationsto ex-plain the hydrazine accumulation behaviour or the possibility to haveNOasintermediateinreactionsinvolvinghydroxylamine(the approach,calculationsandresultsaredetailedinSI).

2.3. Batch tests, kinetic parameters determination

Akinetic model(Table S2 andS3)wasproposed and adapted from van der Star(van der Star et al., 2008b) to obtain the ki-netic parametersofthe reactionsinvolved inthebatch tests. The modelwasimplementedinMatlabR2018bandaimedtominimize theerrorbetweentheexperimentaldataandthesetofdifferential equationsproposed foreach compound.Astheexperiments were performedinbatch,thesetofdifferentialequationswereequalto the resultsof a matrixmultiplication between theproposed sto-ichiometricmatrix andthe processrate matrix(Table S2 andS3, respectively).Astepwiseapproachtoobtainthekineticconstants frommoresimpletomorecomplexbatchtestswasfollowed(Fig. S3). This wascombined withthe useof differentobjective func-tions (Eqs.(S4)–(S7)) toassess ifthe solutionoftheoptimization performedwasindependentoftheobjectivefunctionused(full de-scriptionoftheprocedureisdetailedinSI).

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Table 1

Batch test conditions and speciﬁc rates measured with (if applicable) and without hydroxylamine present. Two duplicates ( n = 2) were performed per condition tested. ∗_– This rate corresponds to hydrazine consumption with hydroxylamine present.

Initial concentrations Speciﬁc rates with NH 2 OH Speciﬁc rates without NH 2 OH Batch NH 4+ NO 2− NH 2 OH N 2 H 4 qNH 4+ ,NH2OH qNO 2−,NH2OH qNH 2 OH ,NH2OH qNH 4+ qNO 2− qN 2 H 4

production qN 2 H 4 consumption mg-N/L mg-N/gVSS/h mg-N/gVSS/h 1 33.4 ± 0.1 16.94 ± 0.02 - - - -46 ± 2 -57 ± 3 - - 2 - - 22.8 ± 0.1 - 18 ± 1 - -76 ± 1 2.9 ± 0.3 - 16 ± 1 -4.3 ± 0.3 3 - - 6.8 ± 0.1 - 9.2 ± 0.6 - -47 ± 2 1.2 ± 0.4 - 7 ± 1 -1.9 ± 0.1 4 1.64 ± 0.03 - 7.7 ± 0.2 - 13 ± 2 - -51 ± 3 2.78 ± 0.04 - 11 ± 1 -3.6 ± 0.1 5 2.4 ± 0.3 - 7.3 ± 0.1 - 12.3 ± 0.5 - -47 ± 1 1.8 ± 0.2 - 5 ± 1 -2.3 ± 0.1 6 29 ± 2 - 21.9 ± 0.8 - 17 ± 2 - -75 ± 1 3.7 ± 0.4 - 32 ± 2 -10 ± 1 7 1.1 ± 0.7 - 4.3 ± 0.1 - - - 3.8 ± 0.1 - - -6.3 ± 0.1 8 - 19.9 ± 0.3 20.1 ± 0.7 - 6.0 ± 0.8 -7.4 ± 0.9 -42 ± 6 -9 ± 1 -7.4 ± 0.9 5.9 ± 0.6 -3 ± 1 9 - 6.89 ± 0.04 7.2 ± 0.6 - 4.7 ± 0.4 -11.0 ± 0.2 -37 ± 2 -5.3 ± 0.9 -11.0 ± 0.2 2.1 ± 0.2 -0.62 ± 0.02 10 84 ± 2 19 ± 3 30 ± 1 - -26 ± 4 -22 ± 2 -113 ± 4 -19 ± 7 -31 ± 2 15 ± 5 -26 11 52 ± 4 18.8 ± 0.1 8.7 ± 0.4 - -31.5 ± 0.9 -17 ± 1 -39 ± 2 -26 -33 ± 1 29 ± 2 -9.8 ± 0.4 12 36 ± 4 10.79 ± 0.01 4.8 ± 0.1 - -42 ± 2 -18.9 ± 0.4 -30.9 ± 0.2 -8.7 ± 0.4 -30.8 ± 0.3 33 ± 1 -6.6 ± 0.6 13 1.5 ± 0.3 - 8.09 ± 0.01 4.90 ± 0.01 33 ± 3 -24 ± 2 ∗ _{-60 ± 4} _{4.0 ± 0.1} _- _- _{-6.4 ± 0.2} Table 2

Reactions involved in hydroxylamine and hydrazine disproportionation. e −_{refers to electron.}_G0 _{R refers to standard Gibbs free energy calculated at 25 °C, 1atm and 1M in} kJ/mol referred to mol of hydroxylamine, ( ∗_{) mol NH}₄+ , ( ∗∗_{) mol N}_{2 H}_{4 or (}∗∗∗_{) mol of NO reacting. Reactions 3, 6, 7 10 and 11 as proposed in the model of van der Star} ( van der Star et al., 2008b ). Half reactions were derived based on N, O, H and charge balances as in ( Kleerebezem and Van Loosdrecht, 2010 ).

Number Name Comments Reaction G 0 R (kJ/mol)

Hydroxylamine disproportionation

1 Hydrazine production (e −_acceptor) _{Half reaction} _N_H_{2 OH + H}+ + e −_{→ 0 . 5 N}_{2 H}_{4 + H}_{2 O} _-149.9 2 Hydrazine production (e −_donor) _{Half reaction} _NH+

4 → 0 . 5 N 2 H 4 + 2 H + + e − _143.3(∗₎

3 Hydrazine production in hydroxylamine

disproportionation

(sum of 1 & 2) N H +

4 + N H 2 OH → N 2 H 4 + H 2 O + H + -6.6

4 Hydrazine consumption (e −_acceptor) _{Half reaction} _N_H₂_{OH + 3 H}+_{+ 2 e}− _{→ NH}+

4 + H 2 O -293.2 5 Hydrazine consumption (e −_donor) _{Half reaction} _N₂_H₄_{→ N}₂_{+ 4 H}+ + 4 e − _-127.8(∗∗₎

6 Hydrazine consumption in hydroxylamine

disproportionation

(sum of 2 ∗_{4 & 5)} ₂_{N H}₂_{OH + N}₂_H₄_{+ 2 H}+ → 2 NH +

4 + N 2 + 2 H 2 O -357.1

7 Hydroxylamine disproportionation (sum of 3 & 6) 3 N H 2 OH + H + → NH 4+ + N 2 + 3 H 2 O -240.3

Hydrazine disproportionation

8 Hydrazine disproportionation (e −_acceptor) _{Half reaction} _N_{2 H}_{4 + 4 H}+ + 2 e − _{→ 2 NH}+

4 -286.6( ∗∗)

9 Hydrazine disproportionation (e −_donor) _{Half reaction} _N_{2 H}_{4 → 4 H}+ + 4 e −_{+ N}₂ _{-127.8 (}∗∗₎

10 Hydrazine disproportionation (sum of 2 ∗_{8 & 9)} ₃_N₂_H₄_{+ 4 H}+ → 4 NH +

4 + N 2 -233.6 ( ∗∗)

11 Hydrazine consumption (option 2 for hydrazine consumption)

N2 H 4 + H + + H _{2 O → N H}+

4 + N H 2 OH 6.6 ( ∗∗)

Hydroxylamine disproportionation via NO, modiﬁed reactions involving hydroxylamine

12 Hydrazine production via NO (e −_donor) _{Half reaction} _NO_{+ 4 H}+ + 4 e −_{→ 0 . 5 N}_{2 H}_{4 + H}_{2 O} _-259.9(∗∗∗₎ 13 Hydrazine production via NO (e −_acceptor) _{Half reaction} _N_H_{2 OH → NO + 3 H}+_{+ 3 e}− ₁₁₀

14 Overall reaction hydrazine production (e −

acceptor)

(Sum of 11 &12), same as 1 N H 2 OH + H + + e −→ 0 . 5 N 2 H 4 + H 2 O -149.9

15 Hydrazine consumption via NO (e −_donor) _{Half reaction} _NO_{+ 6 H}+ + 5 e −_{→ NH}+

4 + H 2 O -403.2( ∗∗∗₎ 16 Hydrazine consumption via NO (e −_acceptor) _{Half reaction} _N_H_{2 OH → NO + 3 H}+ + 3 e − ₁₁₀

17 Overall reaction hydrazine consumption (e −

acceptor)

(Sum of 14 & 15), same as 4 N H 2 OH + 3 H + + 2 e − → NH 4+ + H 2 O -293.2

2.4. Reactor operation - continuous long-term study

One litre of biomass from the same 10L MBR used in batch testsanddescribedinSIwasusedasinoculum fora2LMBR re-actor (Fig. S4). The HRTwaskept at2.3±0.2 dduring thewhole operation using a custom-made ultraﬁltration membrane unit (VanDerStaretal., 2008a).An SRTof8.7±1.0d wasmaintained throughouttheoperationperiodbywithdrawingthedesired reac-tor contentper day. Temperaturewascontrolled at30°C withan externaljacket,andthereactorwasstirredat170rpm.Thereactor pHwascontrolledat7.04±0.04witha53g/LNaHCO3 solution.

When hydroxylaminewascontinuouslyfed (Phase II,days 38-54inTableS4),twobottleswereusedtoavoidhydroxylamine

re-actionwithanyofthemineralmediumcomponents.Thetotal vol-umetricmineralmediumloadsweremaintainedbycorrecting me-diapreparation.

Samples were collected daily from the eﬄuent line and cen-trifuged,supernatantwascollectedforfurtheranalysisofthe dis-solvednitrogencompounds(seeSI)andtreatedwithsulfamicacid ifneeded.Thebiomasspelletswerekeptat-80°C.Afreshsample fromthereactorwasusedtomonitordailyopticaldensity.During reactor operation volatile suspended solids(VSS) were measured periodically, andthe speciﬁc correlation betweenOD660 andVSS

wasusedforcalculations(Fig.S5).DNAsamplesfromdays23,33, 38,40,45,50and55wereanalysedatNovogenfor16SrRNA am-pliconsequencing(seeSI).

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Fig. 1. Dynamics of nitrogen compounds during anammox batch tests with different combinations of substrates: (A) hydroxylamine (Test 2 in Table 1 ) (note that ammonium and hydrazine are represented on the right axis), (B) hydrazine (Test 7 in Table 1 ),(C) hydroxylamine and nitrite (Test 8 in Table 1 ), (D) hydroxylamine, nitrite and ammonium (Test 11 in Table 1 ) (note that ammonium is represented on the right axis). Error bars represent the standard deviation between duplicates.

3. Results&discussion

3.1. Short term anammox metabolism with hydroxylamine as substrate

Anammoxbacteriaprimarily convertammoniumandnitriteto dinitrogengas,yetarealsocapabletometabolizeothersubstrates, suchashydroxylamine,hydrazineandorganiccarbon(Jettenetal., 1998).Inbiofilmsystems,hydroxylaminecanleakfromAOB com-munities(Liuetal.,2017;Suetal.,2019;YangandAlleman,1992), diffusethroughthe biofilm (Sabbaetal., 2015),andreach anam-mox bacteria inanoxic layers. Batch tests to evaluate the capac-ityof anammox bacteria to metabolize hydroxylamine were per-formedby supplying hydroxylaminetogether withdifferent com-binationofsubstratestoa Ca. Kueneniastuttgartiensisenrichment. Theaim wasto investigate ifdifferent combinationofsubstrates impacted the conversion dynamics of the nitrogen species. Hy-droxylamineconcentrationsusedinbatcharehigherthanthoseto whichanammox bacteriamight be exposed innature (i.e. values of0.006-1mg-N/L hydroxylaminehavebeenreportedindifferent nitrificationsystems(Liuetal.,2017;Suetal.,2019;Yangand Alle-man,1992),amongothers),butwereneededtobeableto investi-gatetheconversions.

Trends in nitrogen compounds consumption and production wereindependentoftheinitialconcentrations(Table1).Therefore, only tests with higher initial concentrations are discussed here (Fig.1),unlessdifferentlystated(Fig.S6). Theimpactofsubstrate combinationson hydroxylamine metabolism will be discussed in thepresent section, whereas the impacton hydrazine accumula-tionwillbediscussedfurtherinthenextsection.Positivecontrols toassessanammoxactivitywithammoniumandnitrite(Fig.S7A) wereincluded.Datafromthepositivecontrolsindicatedthatonce nitrite was consumed, no signiﬁcant changes in the ammonium concentrationswere detected. Denitrifyingactivity wasruled out by providing nitrite as substrate(Fig. S7D) to the anammox

en-richmentculture.Abioticcontrolswithboiledbiomassandallthe substratesusedinthebatchtestwereperformedanddidnotshow signiﬁcantactivitycomparedtothebiologicalrates;forexamplean increaseofca.0.1mg-N/L ofammoniumwasdetected inthe abi-oticcontrolafterca.7h(Fig.S7CandE)comparedto5mg-N/Lof ammoniumproducedin2hinbiologicaltests(Fig.1A).

3.1.1. Ammonium production occurs from both hydroxylamine and hydrazine

Whenhydroxylaminewasaddedastheonlysubstrate(Batches 2-5,Table1),hydrazinedidaccumulate,asexpectedfromprevious studies(VanDeGraafetal.,1997;VanDerStaretal.,2010). How-ever,twodistinct ammoniumproductioneventsoccurred(Fig.1A and Fig. S6). The ﬁrst ammonium production startedas soon as hydroxylaminewasadded(ca.0-2.4hinFig.1A,seealso Fig.S6). When the hydroxylamine concentration became low, hydrazine startedto accumulate (around1.6hin Fig.1A, afterca.15-25min in Fig. S6, withlower initial hydroxylamineconcentration). Once hydroxylaminewasdepleted,asecondammoniumproduction pe-riod started, correlating withthedecrease ofhydrazine (fromca. 2.4h onwards inFig. 1A, see alsoFig. S6). Thus, ammonium was produced both with hydroxylamine and hydrazine (Fig. 1A), ap-parently,withpreferenceforutilizationofhydroxylamineover hy-drazine(Fig.S7B).

Theinitial productionofammoniumfromhydroxylamineisin accordance with the hydroxylamine disproportionation reactions (reaction 7,Table 2) proposed by vande Staretal.(vander Star etal.,2008b).Hydroxylaminedisproportionationoccursviatwo in-termediatereactions (reaction3and6,inTable2).Theimbalance ofreactions 3 and6resulted intheobserved hydrazine accumu-lation(seeSection 3.1.4forfurtherdetails).Theexperimental sto-ichiometricratioofconsumedhydroxylamineperammonium pro-ducedwas 3.8mol/mol, which is quiteclose to the theoretically expectedof3forhydroxylaminedisproportionation(reaction7in

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Thesecondammoniumproductionfromhydrazinewasnot ob-served by van der Star et al.(van der Star et al., 2008b), most likelyobscured bythe highinitial ammoniumconcentration used in the tests (2-8 mM, 28-112 mg-N/L). The consumption of hy-drazinewasproposedtofollowtwopossiblereactions(reaction10 or 11, Table 2) (van der Staret al., 2008b). In thepresent study, once hydroxylaminewastotallyconsumed,ca.1.4mM of ammo-niumwereproducedpermMofhydrazineconsumed,closetothe 1.3theoreticalratioofhydrazinedisproportionationviareaction10 inTable1(Fig.1A).Furthermore,hydrazinedisproportionationvia reaction 10 was conﬁrmed by performing a batch test with hy-drazineastheonlysubstrate(Fig.1B,batch 7inTable1).1.2mM mole ofammonium were produced per mM ofhydrazine, which isinagreementwiththeproposedstoichiometryofhydrazine dis-proportionation(reaction 10,Table2)producing1.3molesof am-moniumper mol ofhydrazine.Similar stoichiometriesfor ammo-niumproducedperhydrazineconsumedwerepreviouslyshownin abatchtestperformedwithonlyhydrazinepresent,butno mech-anismswereproposed(VanDeGraafetal.,1997).

Basedontheexperimentalstoichiometry,theproductionof am-monium from hydrazine is most likely to result from hydrazine disproportionation(as inreaction10,Table2,seealso Fig.4,and

Section3.1.8foradiscussiononputativeenzymesinvolved). Theoccurrenceofhydrazinedisproportionationthroughoutthe batchtestcontributedtotheammoniumproductionwhen hydrox-ylamineisstillpresent.Multipleconﬁrmationswereobtainedfrom the experimental data sets and the mathematical model. For in-stance,whenhydroxylamineandhydrazinewereprovidedtogether as substrates both were consumed simultaneously (batch 13 in

Table 1and Fig.S7B). Hydroxylaminewasinitially consumed c.a. 2.5 timesfaster than hydrazine(60 ± 4and 24±2 mg-N/gVSS/h, respectively) anda transient slowdown on hydrazine consump-tioncould beobserved.Furthermore,theproposedmathematical modelwiththeparametersobtained(see Section3.1.9forfurther details)wasusedtocompute theratesofreaction3,6and7 dur-ingabatchtestswithhydroxylamineassubstrate.Thesimulations indicatethathydrazinedisproportionationisoccurringthroughout thebatch test,even ifatone orderofmagnitudelowerratethan hydroxylaminedisproportionation(seeFig.S16).

Overall, two ammonium production events were observed in the samebatch testwithonlyhydroxylamineassubstrate(test2 to 4 in Table 1). When hydroxylamine ispresent, hydroxylamine disproportionation to ammonium anddinitrogen gas via reaction 7isthedominantprocess(Table2)withhydrazine disproportion-ation takingplaceatone order ofmagnitudelower rate(seeFig. S16).Oncehydroxylamineisconsumed,theaccumulatedhydrazine isdisproportionatedtoammoniumanddinitrogengasviareaction 10(Table2).

3.1.2. Ammonium produced from hydroxylamine and hydrazine is used to consume nitrite

Tofurtheranalysetheammoniumproductioncapacityof anam-mox bacteriafromeitherhydroxylamineorhydrazine, batch tests 8and9(Table1)wereperformed.Hydroxylamineandnitritewere dosed simultaneously to assess ifthe ammonium production ca-pacity from hydroxylamine and hydrazine could support nitrite consumption.Asexpected,theammoniumproducedfrom hydrox-ylamine was usedto consumenitrite (Fig. 1C). Aslight transient accumulationof hydrazineandammonium wasmeasured.Nitrite consumptionstoppedassoonasalltheammoniumproducedfrom hydroxylamineandhydrazinewasconsumed(Fig.1CorFig.S7F). Furthermore,theca.20 mg-N/Lhydroxylamineconsumed viathe disproportionationreaction(reaction7,Table1),wouldleadtoca. 6.6mg-N/LNH4+ whichcouldbeconsumedvianormalanammox

metabolism. Ifwe assume a stoichiometry closeto that reported by Lotti etal.(Lotti etal., 2014) of1.146mol NO2− mol NH4+,a

maximumconsumption of7.6mg-N/L nitrite could be converted, which is close to the experimentally observed of 6.4 mg-N/L of nitrite consumed.These observations,together with thefact that in the rest of tests(except tests10-12 in Table 1, that also had nitrite)ammoniumaccumulatedinstead ofbeingconsumed, con-ﬁrmedthatthesubstrateconsumedtogetherwithnitritewas am-monium. The transient accumulation of ammonium with nitrite was shown by Hu and coworkers (Hu et al., 2011), however no hydrazineaccumulationwasdescribedinthosetests.Alsothe hy-droxylamineconversionsloweddownwithtime,maybeduetothe higherconcentrationsusedintheirtests.

Theseresultsfurtherexpandthemetabolicversatilityof anam-mox bacteria: if hydroxylamine is present with nitrite only (i.e.

withoutNH4+),anammoxbacteria cangenerateammonium from

hydroxylamine and consume nitrite via the canonical anammox conversion.These resultsshow a potential role of hydroxylamine in Ca. Kuenenia stuttgartinesis metabolism, in contrast to the prior unique implication ofhydroxylamine in Ca. Brocadia sinica (Oshiki etal., 2016). Thissituation mightoccur in partial nitrita-tion/anammox(PN/A) systems whenammonium isfullydepleted whileresidual,lowconcentrationsofhydroxylaminemightbestill present,e.g.inbioﬁlmsduetogradientsbetweenmicrocolonies.

3.1.3. Hydroxylamine is consumed simultaneously with nitrite, but faster

Batch testswere performedto assess the impact of hydroxy-lamine when both nitrite and ammonium were present(batches 10,11, 12,Table1). Thissubstratescombinationis likelytooccur inPN/Abioﬁlms,althoughhydroxylamineconcentrationsmightbe lowerthanthoseusedhere. Hydroxylaminewasconsumed simul-taneously with nitrite (Fig. 1D). The speciﬁc hydroxylamine con-sumptionrate (qNH2OH) was1.6 to 5 times higher than that of

nitrite (qNO2−), depending on the initial hydroxylamine

concen-tration.Assoonasallhydroxylaminewasdepleted,a50%increase ofnitrite consumptionratewasmeasured (Table1). Interestingly, evenafterhydroxylaminedepletion, nitrite consumptionrates re-mainedlowerthanthemaximalratemeasuredinpositivecontrols, performedwithnitriteandammonium,usualanammoxsubstrates (batch1,Table1).Thus,theseresultssuggestthathydroxylamineis consumedsimultaneouslywithnitrite,butfasterthannitrite. Hy-droxylaminepresencetogether withnitrite andammonium hada putative toxic or partially irreversible effect on nitrite consump-tionrates,aswhenhydroxylaminewasfullyconsumednitrite con-sumptionratesdidnotreachthelevelsofthepositivecontrols.

Withinthe range of concentrationstested, the hydroxylamine consumption ratelinearlydepended onthe initial hydroxylamine concentration(Fig.2A),andwasnotaffectedbythecombinationof availablesubstrates.Thelinearconsumptionofhydroxylamine ob-servedis consistentwithpreviously reportedmeasurements with ammoniumonly(vanderStaretal.,2008b).Inbiologicalsystems, Monod-likekineticsareusually observedforsubstrates consump-tion.ForMonod kineticstobe linear, thesubstrateconcentration hastobe smallerthanthehalfsaturationcoeﬃcient.Thus,inthe presentstudy, the hydroxylamine half saturation constant would then be unexpectedly high for suspended cells (ca. _> 22 mg-N-NH2OH/L, Fig.2A),asforexamplethehalfsaturationconstantfor

nitrite ina similar culturewas35 μg-N/L(Lottiet al., 2014). An-otherputativeexplanationfortheobservedlinearityisthepassive transport of hydroxylamine over the membrane. Hydroxylamine hasapKa of5.9at25°C(Haynes,2014),thus itismostly unpro-tonatedunder thetestedconditions(pH8 and30°C inthebatch tests).Asaresult,passivediffusionthroughthemembrane,strictly dependingonthedifferencebetweenthebulkandcell concentra-tionofthesubstrate,islikely(Albertsetal.,2007).

Ultimately, the presented results further expand the known metabolicversatilityofanammoxbacteria.Hydroxylamineand

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hy-Fig. 2. Impact of initial NH 2 OH concentration [NH 2 OH (mg-N/L) in A] or specific initial hydroxylamine concentration [NH 2 OH/C x (mg-N/gVSS) in B and C] to: (A) initial specific hydroxylamine rate [qNH 2 OH (mg-N/gVSS/L)], (B) time to reach the hydrazine peak [Time to peak (h)] and (C) maximum measured hydrazine concentration [N 2 H 4 peak (mg-N/L)]. Cx stands for biomass concentration (gVSS/L). Batch tests were performed with different combination of substrates: (i) NH 4 + , NO ₂−_{, NH}_{2 OH (squares), (ii)} NH 4 + , NH 2 OH (empty circles), (iii) NH 2 OH (filled circles), (iv) NO 2 −, NH 2 OH (triangles). Linear dependencies between parameters are shown in A and B, independently of the combination of substrates used. Batch tests were ammonium was present (squares and empty circle) had higher N 2 H 4 peak than when no ammonium was present (filled circles and triangles). Notice the Y axes are different in each figure and X axis scale is different in Fig. 2 A. Error bars represent the standard deviation between biological duplicates.

drazine disproportionation were proven to occur simultaneously and,ifavailable,hydroxylaminewasshowntobeconsumed simul-taneouslyandfasterthannitriteassubstrate.

3.1.4. Hydroxylamine disproportionation controls hydrazine accumulation

Todate,hydrazineaccumulationhasonlybeenreportedinthe presenceof hydroxylamine (e.g.(Jetten etal., 1998; Kartal etal., 2011;Van DeGraafetal., 1997)).However, hydrazineisnot usu-allyanalysed in anammox systems, thus its actual concentration isunknown.Tounderstandthemechanismsunderlyinghydrazine turnover,hydrazineconcentration wasmeasured duringall batch tests(Table1).Hydrazinetransiently accumulatedwhen hydroxy-laminewasaddedassubstrate(Batches2-6and8-12inTable1), andthetime ofthe hydrazinepeak dependedon theinitial spe-ciﬁc hydroxylamine concentration (mg-N/gVSS, Fig. 2B), consis-tentlywithvanderStar(vanderStaretal.,2008b).

Hydrazine transient accumulation can be explained with hy-drazinedisproportionation(reaction7,Table2).The disproportion-ationistheadditionoftworeactions(reactions3and6,Table2). Hydrazinestartsaccumulatingwhenhydroxylamineconcentration islow(ca. < 5mg-N/LinFig.1A).Hydroxylamineconcentration af-fectsbothhydrazineproduction(reaction3,Table2)andhydrazine consumption(reaction6,Table2),buttwomolesofhydroxylamine are needed for hydrazine consumption as opposed to one mole neededforits production.Thus, alower concentrationof hydrox-ylaminedecreasesthehydrazineconsumption ratemorethanthe hydrazineproductionrate, leadingtotransienthydrazine accumu-lation.Thiswasfurthersupportedbythemathematicalmodel(Fig. S16):thehydrazineconsumptionrate(viareaction6inTable2)is alwayslowerthanthehydrazineproductionrate(viareaction3in

Table2),resultinginhydrazineaccumulation.

Originally, theaccumulationofhydrazineuponadditionof hy-droxylamine was ascribed to the inhibition of HDH, the enzyme responsiblefortheconversionofhydrazinetodinitrogengas.This hypothesiswasbasedontheobservedin vitro inhibitionofHDHby NOandhydroxylamine(Maalcke etal., 2016; VanDe Graafetal., 1997). However, our results show that HDH inhibition cannot be the explanation of hydrazineaccumulation, as hydrazinedid not accumulatedirectlyafterhydroxylamineadditionathighinitial hy-droxylamine concentrations (ca. 20 mg-N/L Fig. 1A). Instead, the hydrazinepeakoccurredaftertheconsumptionofmorethanca.15 mg-N/Lofhydroxylamine,whenhydroxylamineconcentrationwas

< 5 mg-N/L. This delaywas also observed by van der Star et al. (vander Staret al., 2008b), who initially proposed that HDH

in-hibition could not be thecause forhydrazine accumulation.This iscontradictory withtheresultsofMaalcke etal.(Maalcke etal., 2016) that showed that HDH wasinhibited by NO and hydroxy-lamineinvitro.Similarly,Huandco-workersdidnotobserveany signofHDHinhibitionwhenNOwasfedcontinuouslytoan anam-mox culture(Hu etal., 2019). Overall, theHDH inhibition by hy-droxylamine(andNO)demonstrated in vitro (Maalckeetal.,2016) wasnotobservedtooccur in vivo ,meaninganotherprocess under-liesthehydrazineaccumulation.Instead,theimbalanceofthe hy-drazineproductionrateandconsumptionrate(reaction3and6in

Table1)duringhydroxylaminedisproportionationistheproposed causeofhydrazineaccumulation.

3.1.5. Hydrazine accumulation depends on the available substrates

The wide range of combinations of added substrates used in thisresearch showedthat hydrazineaccumulationisstrongly im-pacted by the used substrate combination (Fig. 2C). Speciﬁcally, the accumulationofhydrazine washigherwhen ammoniumwas present,whilethepresenceofnitriteresultedinlower accumula-tions (Fig.2C). Previousresearch onhydrazineaccumulationused only ammonium and hydroxylamine as substrate (van der Star etal.,2008b).

This can also be explained by the imbalance of reactions 3 and6duringhydroxylaminedisproportionation.Forthehydrazine production during hydroxylamine disproportionation, ammonium is consumed (reaction 3, Table 2), thus higher concentration of ammonium would favour hydrazine production. Contrarily, hy-drazineconsumptionresultsinammoniumproduction(reaction6,

Table2),thusthepresenceofammoniumwouldresultinthis re-actionbeinglessthermodynamicallyfavourable(seeSection3.1.6). On the other hand, nitrite decreased the hydrazine peak (Fig.2C). Whennitriteandhydroxylaminearepresentastheonly substrates,nitrite isconsumedwiththe ammoniumthat isbeing produced from eitherhydroxylamine or hydrazine disproportion-ation (Fig. 1C). Thus, nitrite presence decreases the ﬁnal ammo-niumconcentration,favouring hydrazineconsumption (reaction6,

Table2).Whenbothammoniumandnitritewerepresenttogether with hydroxylamine (batches 10,11 and 12, Fig. 1D), ammonium dominatedthepossibleeffectofnitrite,leadingtohighhydrazine accumulations.

Overall,we showedthathydrazineaccumulationoccursinthe presenceofhydroxylamine, andtheaccumulationispromoted by ammoniumandreducedbynitrite.Elucidatingtheroleand occur-renceofhydrazineaccumulationinbiologicalsystemsrequires fur-therfull-scale experimentalconﬁrmation asmeasurements of

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hy-Fig. 3. Thermodynamics of selected reactions during different batch tests (Test 2, 6, 8 and 10 of Table 1 ) (A) ammonium to hydrazine conversion electron donor (reaction 2 in Table 2 ) is the only thermodynamically positive conversion, (B) hydrazine production in hydroxylamine disproportionation (reaction 3 in Table 2 ), (C) hydrazine consumption in hydroxylamine disproportionation (reaction 6 in Table 2 ), (D) NO conversion to hydrazine (reaction 12 in Table 2 ).

drazine duringreactoroperation remain rare.Hydrazine accumu-lationorleakagebyanammoxwouldbeunfavourablefroman en-ergetic pointofview,ashydrazinetransformationtoN2 isone of

the anammox electron sources (see Eq.(3)).However, leakageof hydrazinecanbeapotentialadvantageforanammoxagainstother direct competitors,ashydrazineistoxicto other microbesofthe nitrogen cycle, such asNOB(Yao etal., 2013). Furtherdiscussion on thethermodynamicsofhydrazineaccumulationandthe puta-tiveenzymesinvolvedcanbefoundinSections3.1.6–3.1.8.

3.1.6. Thermodynamics cannot explain hydrazine accumulation

To identify anypossible thermodynamic limitationunderlying the observed hydrazine accumulation, a thermodynamic analysis of the reactions involved(Table 2) wasperformedalong concen-trationproﬁlesduringbatchtests(Figs.S8andS9).

All reactions involvedinhydrazineandhydroxylamine dispro-portionation were thermodynamically favourable, as they had a negativeGibbsenergychangeofreaction(

G0

R),withthe

excep-tionofthehalfreactionofammoniumconversiontohydrazine (re-action 2,Table2). Evenafterthecorrection foractual batch con-ditions, hydrazineproduction fromammoniumhada positive ac-tualGibbsenergychangeofreaction(

G1

R)(Fig.3A).Evenifone

of the half reactions was not thermodynamically favourable (re-action 2), the overall hydrazineproduction (reaction 3) had neg-ativeactualGibbsfree energy.However,thehydrazineproduction step(reaction3;Table2),wasclosetotheequilibrium,andbatch conditionsstrongly impacteditsoverall

G1

R (Fig. 3B). Thus,

hy-drazineproductionthermodynamicsheavily dependonbatch test conditions (Fig. 3A and B). Conversely, the reactions involved in

hydrazine consumption were strongly favourable (Fig. 3C). Con-sequently, the thermodynamic analysis doesnot explain the ob-served accumulation of hydrazine. A kinetic limitation or and enzymatic/biologicalbottleneckimpacting hydrazineconsumption mightbetheexplanation,asalsodiscussedintheprevioussection andasshownbythemathematicalmodel(Fig.S16).

Impact of batch conditions, namely pH, temperature, ammo-niumandhydroxylamineconcentration,onthepotentialhydrazine conversions were also investigated (Fig. S11). Higher hydroxy-lamineandammoniumconcentration(Fig.S11AandB)resultedin amorefavourablehydrazineproduction,inagreementwiththe ex-perimentalresults(Fig.2C).HigherpHwasalsoshowntomake hy-drazineproductionmorethermodynamicallyfavourable(Fig.S11E), whereastemperaturehadlittleimpact overthetestedrange(Fig. S11D).In thisperspective, it is worth noting that the anammox-osome - where hydrazine is being produced - is more acidic than the cytoplasm(Van Der Star etal., 2010; van Niftrik etal., 2004), thusmaking hydrazineproductionlessthermodynamically favourable.

3.1.7. Thermodynamics suggest that NO is an unlikely intermediate in hydroxylamine turnover

Thefeasibility andpotentialoccurrenceof NOasintermediate inthereactionsinvolvinghydroxylamine(reactions12-17;Table2, Fig.S10)wasanalysedbasedonthermodynamic characteristicsof theconversions. Hydroxylamineis hypothesizedtobe ﬁrst trans-formedtoNO,andthenNOistransformedtotheﬁnalproduct.The estimated

G1

RforthetransformationofNOtohydrazineduring

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Fig. 4. Putative enzymes involved in hydroxylamine disproportionation conversions depending on the assumed intermediates: Black lines are conversion where hydrox- ylamine is directly transformed to hydrazine or ammonium, black dashed lines are reactions where NO might be an intermediate. Numbers correspond to reactions in Table 2 , where corresponding Gibbs free energy values can be found. Regular anammox metabolism is represented with grey dashed boxes and arrows. Notice that NO and N 2 H 4 have both solid and dashed line boxes. ∗Anammox genome encodes more than 10 HAO-like proteins, which the function of some of them is still unknown, ∗∗_HZS has been proposed to have hydroxylamine as inner intermediate ( Dietl et al., 2015 ), or in Ca. Brocadia it has been shown to transform hydroxylamine and ammonium to hydrazine ( Oshiki et al., 2016 ; Oshiki et al., 2015 ). Thus, we hypothesise that there is an HZS-like protein able to transform ammonium and hydroxylamine to dinitrogen gas from the more than 10 HAO-like proteins encoded in anammox genome.

unfavourable(positive)dependingontheconditions(Fig.3D; reac-tion12,Table2).As aresult,consideringthepreviouslydiscussed closetoequilibriumreaction3(i.e.hydroxylaminetoNO;Table2), thetransformationofhydroxylamineviaNOwouldhavetwo inter-mediatesteps closeto thermodynamic equilibrium. Forma ther-modynamicpoint ofview theforwardconversionis possible,but highlydependingon the exactbatch test conditions.The experi-mentalevidenceonthepotentialroleofNOasintermediateinthe hydroxylamineconversionwouldstillbeneeded.

Ultimately,itshouldalsobenotedthatabettercharacterization ofhydroxylamine standard Gibbs energy offormation (G°f,NH₂OH)

valueisneededforadeﬁnitivethermodynamicstudy.Inliterature, differentvaluesforG°f ofhydroxylaminecanbefound (TableS1),

whichprofoundlyimpacttheobtainedresults(Fig.S12).

3.1.8. Hydroxylamine and hydrazine disproportionation can be explained by known enzymatic anammox conversions

We hypothesize that disproportionation ofhydroxylamine and hydrazinemightbecatalysedbymultipleenzymes,mostofwhich are alreadycharacterized in anammox bacteria. For instance, hy-drazine production(reaction 3in Table 2) in hydroxylamine dis-proportionationcouldbecatalysedbydifferentcombinationof en-zymes, eitherwith NO as intermediate or not, in hydroxylamine conversions:i)NH2OH isﬁrsttransformed toNO (reaction12,13

andoverall 14)by HOX.Then, NOandNH4+ couldbe reducedto

hydrazine(reaction 2)byHZS(see Fig.4).ii)Alternatively, anam-mox bacteria encode more than 10 anammox HAO-like proteins in the genome, the function of some of them is still unknown. Thus,itcould be thatone oftheHAO-likeproteins in Ca. Kuene-niacould performasimilarconversionastheHZSin Ca. Brocadia (Oshikietal.,2016).Thus,theBrocadia-likeHZStransformsdirectly hydroxylamineandammoniumtohydrazine,andHDHfunnels hy-drazineintodinitrogengas(reaction 1,2leadingtotheoverall re-action3)(seeFig.4).

Hydrazine consumption (reaction 6) in hydroxylamine dispro-portionationcould beacombinationofaHAO-likeprotein reduc-ing NH2OH to ammonium (reaction4) andthe knownactivityof

HDH(reaction5)(seeFig.4).

Hydrazine disproportionation (reaction 10) would need HDH (reaction9) anda dedicatedenzymetoproduceammoniumfrom hydrazine(reaction 8).Anenzyme catalysingthislast conversion, hasnotbeendescribedyetinanammox.

From the overall enzymatic conversions proposed, ammonium production from hydroxylamine and hydrazine have not been shownin anammoxbacteria. However, ammoniumproducing ac-tivity from hydroxylamine has been hypothesized to exist based onmetagenomicdatafromtheanammoxbacterium Ca. Scalindua profunda (vande Vossenbergetal., 2013).Furthermore, hydroxy-lamine conversion toammonium has been shownin ammonium oxidizing bacteria Nitrosomonas (Kostera et al., 2008) and enzy-maticactivityfound inthe dissimilatorynitratereducing bacteria

Nautilia profundicola (Hansonet al.,2013), itis hypothesizedthat thereisanHAO-likeenzymewithanammoniumproducing activ-ityfromhydroxylamine. Nevertheless, tofurther conﬁrm this hy-pothesis,transcriptomicsandproteomicsdatawouldbevaluable.

3.1.9. Kinetic modelling supports the potential impact of hydroxylamine on nitrite metabolism

Theestimationofthekineticparametersforthediscussed reac-tions istheprerequisite fortheir inclusion inmathematical mod-els(Henzeetal.,2000).Experimentalparameterdetermination re-quires highlyprecisemeasurements methods,andusually kinetic modelsareapplied(vanLoosdrechtetal.,2016). Thefactthat hy-droxylamine is consumed via two simultaneous reactions (reac-tions 3 & 6, Table 2), and theneed of both of them to describe hydrazineaccumulation,makestheexperimentaldeterminationof parameters impossible. Instead, a step-wise modelling approach using different optimization functions was used to assess if the

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Table 3

Kinetic parameters determined with the kinetic model. When standard deviation is given, average between the results obtained with different objective functions was performed. When no standard deviation is given, the value selected is the one that resulted in a smaller error between the model and experimental data.

Substrates k 1 K 1,NH 2OH K 1,NH 4+ k 2 K 2,NH 2OH K 2,N 2H 4 k 3 K 3,N 2H 4 k 4 K 4,NH 4+ K 4,NO 2

-mmol/gVSS/h mM mM mmol/gVSS/h mM mM mmol/gVSS/h mM mmol/gVSS/h mM mM

N 2 H 4 0.285 ±0.007 0.030 ±0.003

NH 2 OH 2.8 0.16 0.057 2.8 0.0027 0.56

NO 2 −_{+ NH}₄+ _{4.5 ±0.1} _{0.78 ±0.01 0.03 ±0.04}

NO 2 −+ NH 4 + + NH 2 OH 2.2 1.6 0.004

available data setallows forparameterdetermination (see SI).To thisend,thekineticmodelproposedbyvanderStar(vanderStar etal.,2008b)wasadaptedtotakeintoaccounttheimpactof am-monium (Table S3). The anammox stoichiometry wasalso intro-ducedinthesetofreactionstomodeltheimpactofhydroxylamine additionwithammoniumandnitrite(TableS3).

Two independentoptimizations were performedto determine thekinetic parametersofhydrazineconsumption (k3 andK3,N₂H₄)

andanammox(k4, K4,NO₂-,andK_4,NH

4+).Independentlyoftheused optimizationfunction,asinglesetofparameterswasobtained(Fig. S13AandD, andS14AandD).However, whenhydroxylaminewas the only substrate, the values obtainedfor theremaining kinetic parameters (k1, K1,NH2OH, K1,NH4+,k2, K2,NH2OH, K2,N2H4) depended on the objective function used (Figs.S13B andS14B). The set of parameter values (k1, K1,NH₂OH, K1,NH₄+, k2, K2,NH₂OH, K2,N₂H₄)

re-sultinginthelowestsumofsquarederrorswasselected(Table3), andusedinsubsequentoptimizations.

Next, parameters obtained with control (k4, K4,NO₂-, and K_4,NH

4+), hydrazine(k3 andK3,N2H4) andhydroxylamine tests(k1, K1,NH2OH,K1,NH4+,k2,K2,NH2OH,K2,N2H4)wereusedtosimulatetests whereammonium,nitriteandhydroxylamineweresimultaneously provided.Withoutanyextraoptimizationstep,theparameters pre-viously obtainedwere notabletodescribetheexperimentaldata. Speciﬁcally, the depletion of nitrite was predicted to be faster by the model (Fig. S15A), suggestinga direct impact of hydroxy-lamine on the regular anammox metabolism of nitrite consump-tion. Consequently, parameters affecting the nitrite consumption (k4, K4,NO₂-, andK_4,NH

4+) were optimized using the experimental data when hydroxylamine was present, while the other parame-terswerekeptconstant(Figs.S13C,S14CandS15B).The optimiza-tionresultedinca.a50%decreaseinthespeciﬁcmaximum anam-mox rateconstant (k4),and50% increase inthe nitrite half

satu-rationcoeﬃcient(K4,NO₂−).Consistentlywiththeexperimental

re-sults,theseobservationsfurthersupportthe strongimpactof hy-droxylamineonthenitriteconsumptionbyanammoxbacteria.

In literature, the only available set of parameters for the re-actions involving hydroxylamine (van der Star et al., 2008b) es-timated a maximum rate constant (k) one order of magnitude smallerthantheonesreportedinthepresentstudy(Table3).This could be explained by the useof granularbiomass (van der Star et al., 2008b) instead of planktonic culture asdone here. Part of thebiomassinthegranulescouldbeinactiveleadingtoan appar-ent(slower)rate.Moreover,differencesintheaveragegrowthrates betweenthetwo systemscouldalsocontribute tothe differences inmaximumrateconstants.

3.2. Long term continuous exposure to hydroxylamine reduces the NO 3− production

Thelongtermeffectsofcontinuousexposureofanammox bac-teria to hydroxylamine had not been studied yet. To thisend, a planktonic cultureof Ca. Kueneniastuttgartiensis wasoperatedin continuousmodeformorethan54days(Fig.5A,Fig.S17and Ta-ble S4). After the initial 20 daysof stabilization,two operational phasescanbedistinguished:i)PhaseIwithammoniumandnitrite

(days20-37,TableS4),ii)PhaseIIwithammonium,nitriteand hy-droxylamine(days38-54,TableS4).Tomimictheexpectedlow hy-droxylamineaccumulation byAOB inPN/Aprocesses,asmall hy-droxylamine load(ca. 26 mg-N/L/d)compared to the nitrite load (ca.478mg-N/L/d;TableS4)waschosen.

The addition of hydroxylamine did not impact the microbial community composition as revealed by 16S rRNA amplicon se-quencing(Fig.5B).Therelativeabundanceof Ca. Kuenenia stuttgar-tiensis remained stable at79±4 % during2 complete SRTs with hydroxylaminefeeding.Thegenus Ignavibacterium representedthe mostabundantsidepopulationduringthewholeoperation.These resultsindicatethatsimultaneous consumptionofhydroxylamine, ammonium and nitrite does not impact anammox bacteria, and mightevenrepresentacompetitiveadvantageinbioﬁlmPN/A sys-tems against canonical NOB, often reported to be inhibited by hydroxylamine (Blackburne et al., 2004; Blackburne et al., 2008;

CastignettiandGunner,1982;HaoandChen,1994;Noophanetal., 2004;Wangetal.,2015).

During the whole experiment, hydrazine and hydroxylamine concentrations remained below detection limits (Fig. S17C). As soonashydroxylaminewasfed,a statisticallysigniﬁcant decrease (p ≤ 0.001, Mann-WhitneyRankSumTest) inthenitrate produc-tiontoammonium consumption ratiowasobserved,from0.24± 0.03(PhaseI) to0.17_±0.01 mol-NO3−/mol-NH4+ (Phase II)(Table

S4andFig.5A).Thus,hydroxylaminereducedtheformationof ni-trate in the anammox conversion. It is noteworthy that the re-sponsewasimmediatewithoutanyvisibleadaptationeffect.Based on the anammox biochemistry presented in this study, hydroxy-lamine can be metabolized via two pathways: i) NH2OH is

con-verted toNO andthen further to N2 via theconventional

anam-moxmetabolism in Eqs.(1)–(3),orii)NH2OH is transformed via

hydroxylaminedisproportionationreactions,formingNH4+andN2

as in the batch tests (reaction 7, Table 2). From nitrogen mass balances only,the hydroxylaminepathwaycould not beresolved. Giventhe low hydroxylamineload compared to nitrite, hydroxy-laminedisproportionation wouldhaveresulted inca.8 mg-N/L/d ofextraammonium.Suchlowconcentrationswouldbemaskedby thehighresidualammonium concentration inthe reactor. Never-theless,inastudyfromvanDeGraafandcolleagues(VanDeGraaf etal.,1997)theadditionof15_NH

2OHtogetherwithunlabelled

am-moniumandnitrite leadto30_N

2 production,mostlikelyresulting

fromthereactionof15_NH

4+-producedfromhydroxylamine-and 15_NH

2OHitself.Thus,basedonthelatterstudyandthebatchtests

performedhere, hydroxylamine disproportionation islikely to be thedominantpathway.

Recently,anammoxbacteria were alsoshowntogrowon only NO andammonium,withno nitrateproduction(Huetal., 2019). Theseresultschallengedthecommonassumptionthat nitrite oxi-dationtonitrateisneededtoprovidetheelectronstoreduceCO2

forbiomass synthesis.Instead, thenewhypothesis proposed that the high energyelectrons produced during hydrazine conversion to N2 are morelikely those used in anabolism (Hu et al., 2019).

Similarly,inthe presentstudya cleardecreaseinnitrate produc-tion wasobserved withcontinuoushydroxylamine feeding. Inde-pendentofthepathway, hydroxylamineconversionreleases extra

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Fig. 5. Reactor operation dynamics: (A) stochiometric ratios during reactor operation without (white background) and with hydroxylamine load (grey area). (B) Microbial relative abundance based on 16S rRNA amplicon sequencing of reactor samples at different operational days.

highenergycontentelectrons reducingtheneedfornitrite oxida-tiontonitrate.

Thisstudyfurtherextendsourknowledgeofthemetabolic ver-satilityofanammoxbacteria,demonstratingthesimultaneous con-sumptionofammonium, nitrite andhydroxylamineby anammox bacteria. Furthermore, the reactions involved in hydroxylamine metabolismwerefurthercharacterizedusingbatchtests.This char-acterizationresultedinthefollowingﬁndings:(i) ammoniumcan beproducedfromeitherhydroxylamineorhydrazine;and(ii)the co-metabolization ofother substrates impacts hydrazine accumu-lation.In addition, hydrazine accumulation was analysed from a kinetics and thermodynamics point of view, further conﬁrming thathydrazineaccumulationisgovernedbythereactionsinvolved inhydroxylaminedisproportionation,ratherthaninhibition of hy-drazinedehydrogenase.

Overall, this work highlights the huge metabolic versatility of anammoxbacteria.Thisuniqueabilitytouseabroadrangeof sub-stratesrepresentsaclear competitiveadvantage likelyunderlying theabilityofanammoxtothriveindifferentenvironments.For ex-ample,hydroxylamineisknowntobetoxicforNOB,directly com-peting with anammox for nitrite (Castignetti and Gunner, 1982;

Stüvenetal., 1992; Yang andAlleman, 1992). From an engineer-ingpointofview,thiskindofcompetitiveadvantagecanbeuseful in systems like partial nitritation anammox,where NOB are not desired. For example, hydroxylamine external addition has been testedto obtain a successfulpartial nitritation anammox process inlaboratoryconditions(Wangetal.,2015).

Finally, the presentwork sets thebasis to further understand hydroxylaminemetabolismbyanammoxbacteria.Nextstepscould be directed to further conﬁrm the pathway followed by hydrox-ylamine in continuousoperation witheither 15_N_labelling _or

in-creased loads. Doing similar experiments with other anammox bacteria species(i.e. Ca. Brocadia) would also help to assess pu-tativedifferencesbetweenanammoxspeciesmetabolism. Compar-ativetranscriptomicorproteomic datawouldprovidemore infor-mationintheenzymesinvolvedintheprocess.Overall, intermedi-atesofthenitrogencycleareoverlookedandnotstudiedindepth, andcould be asource fornot recognized conversions or interac-tionsinmicrobialcommunities.Understandingsuchinteractionsis crucialforimprovingitsimplementationastechnology.

4. Conclusions

The combinationofbatchtests, continuousfeeding, thermody-namics analysisand modellingallowed to elucidate more details aboutthehydroxylaminemetabolismofanammoxbacteria,

• Ammoniumcanbeproducedfrombothhydroxylamineand hy-drazine.Ifonlynitriteandhydroxylamineareavailable,

ammo-niumcanbeproducedfromhydroxylamineandusedfornitrite consumption.

• When hydroxylamine, ammonium and nitrite are present to-gether inanammoxbatch tests, hydroxylamineandnitrite are consumed simultaneously, with hydroxylamine consumption beingfasterthannitriteconsumption.

• Hydrazine accumulation only occurs when hydroxylamine is presentandseems tobe due toa biological imbalancerather thana thermodynamic limitationorenzymaticinhibition. The extentofhydrazineaccumulationdependsonthecombination of substrates provided, i.e. promoted by ammonium and re-ducedbynitrite.

• Anammoxbacteria cangrow simultaneously withammonium, nitriteandhydroxylamine,reducingthenitrateproduction.

• Anammoxmicrobial population is not impactedby long term feedingofhydroxylamine.

• Hydroxylamine,ifavailable intheenvironment, mightplayan importantyetoverlookedroleinthemetabolismof Ca. Kuene-niastuttgartiensis.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompeting ﬁnan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto inﬂuencetheworkreportedinthispaper.

Acknowledgments

AuthorswouldliketoacknowledgeBenAbbasforthehelpwith DNA extractions and 16S amplicon sequencing. Gerben Stouten for the reactor set-up and discussion. Robbert Kleerebezem and ChristopherLawsonfordiscussion.

This research wasﬁnancially supported by the SIAM Gravita-tionGrant024.002.002,theNetherlandsOrganizationforScientiﬁc Researchandby theDutchTechnology Foundation(STW-Simon StevinMeester 2013)andby theSpanishMinisteriodeEconomía, Industria y Competitividad(MINECO), Agencia Estatal de Investi-gación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, EU),CTQ2017-82404-R.MicheleLaureniwassupportedbyaMarie Skłodowska-CurieIndividualFellowship(grantagreement752992), andaVENIgrantfromtheDutchResearchCouncil(NWO)(project numberVI.Veni.192.252).

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.watres.2020.116188.

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