Ammonia determines transcriptional profile of microorganisms in anaerobic digestion

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Ammonia

determines

transcriptional

profile

of

microorganisms

in

anaerobic

digestion

Nan

Zhang

_,

_Huijuan

_Peng

_,

_Yong

_Li

_,

_Wenxiu

_Yang

_,

Yuneng

Zou

_,

_Huiguo

_Duan

a_{NeijiangNormalUniversity,CollegeofLifeSciences,Neijiang,China}

b_{DepartmentofEducation,KeyLaboratoryofRegionalCharacteristicAgriculturalResources,Neijiang,China}

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Received25September2017 Accepted13April2018 Availableonline21May2018 AssociateEditor:ValeriaOliveira

Keywords: Anaerobicdigestion Ammonia Pathway Geneexpression Methanogenesis

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Anaerobicdigestionisimportantforthemanagementoflivestockmanurewithhigh ammo-nialevel.Althoughammoniaeffectsonanaerobicdigestionhavebeencomprehensively studied,themolecularmechanismunderlyingammoniainhibitionstillremainselusive. Inthisstudy,basedonmetatranscriptomicanalysis,thetranscriptionalprofileofmicrobial communityinanaerobicdigestionunderlow(1500mgL−1)andhighNH4+(5000mgL−1)

con-centrations,respectively,wererevealed.TheresultsshowedthathighNH4+concentrations

significantlyinhibitedmethaneproductionbutfacilitated theaccumulations ofvolatile fattyacids.Theexpressionofmethanogenicpathwaywassignificantlyinhibitedbyhigh NH4+concentrationbutmostoftheotherpathwayswerenotsignificantlyaffected.

Further-more,theexpressionsofmethanogenicgeneswhichencodeacetyl-CoAdecarbonylaseand methyl-coenzymeMreductaseweresignificantlyinhibitedbyhighNH4+concentration.The

inhibitionoftheco-expressionsofthegeneswhichencodeacetyl-CoAdecarbonylasewas observed.Somegenesinvolvedinthepathwaysofaminoacyl-tRNAbiosynthesisand ribo-somewerehighlyexpressedunderhighNH4+concentration.Consequently,theammonia

inhibitiononanaerobicdigestionmainlyfocusedonmethanogenicprocessbysuppressing theexpressionsofgeneswhichencodeacetyl-CoAdecarbonylaseandmethyl-coenzymeM reductase.Thisstudyimprovedtheaccuracyanddepthofunderstandingammonia inhibi-tiononanaerobicdigestion.

©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Anaerobicdigestion(AD)isapromisingtechnologyinthefield ofwaste treatmentandrenewableenergyproduction. Live-stockmanurehasbeenprocessedincreasinglybyADtoreduce

∗ _{Correspondingauthorat:No.705,DongtongRoad,DongxingDistrict,Neijiang641100,China.}

E-mail:duanhuiguo6@163.com(H.Duan).

pathogens and to generate bioenergy such as methane.1,2

Thus, the improvementofstability and efficiency ofADis crucialforthecomprehensiveapplicationofthistechnology. Ammonia concentrationisoneofcrucialfactors regulating ADstability.3_{Optimalammoniaconcentrationsprovide}

suffi-cientbuffercapacityandnutrientformicrobialgrowth,which improves the AD stability and efficiency. However, low or

https://doi.org/10.1016/j.bjm.2018.04.008

1517-8382/©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

excessiveammoniaconcentrationsusuallyresultinthe fail-ure of AD. Itis reported that low ammonia concentration (<500mgL−1) decreases methane production, biomass and theaceticlasticmethanogenic activity.4 _{Thehigh ammonia}

(>4000mgL−1)resultsinthe inhibitionofmicrobial activity andaccumulationofvolatilefattyacids,whichfinallycauses decreased stability and even failure ofAD.3–6 _{Due to high}

proteincontentinmanure,thereareusuallyhighammonia concentrationsintheADofmanure.3 _{Thus,ADoflivestock}

manurehastoconfronttheinhibitionfromhighammonia.It isnecessarytorevealthemechanismsunderlyingammonia inhibitionontheADprocess.

Temperatureisconsideredasaprominentfactor regulat-ing ammonia toxicityon AD process.3,7,8 _{Thethermophilic}

temperaturescomparedtomesophilictemperaturesusually causehigherammoniatoxicity.7,8_{Thethermophilic}

tempera-turesenhancemetabolicactivitiesofmicroorganisms,which strengthenshydrolysisofsubstratesincludingproteins.This process increases ammonia concentrationin the slurry,so that ammonia toxicity is undoubtedly strengthened.Thus, undermesophilictemperatures,theammoniatoxicitymore depended on the initial ammonia concentration, which facilitates the understanding of the relationship between ammonia concentration and ammonia toxicity. Different methanogens are distinguishably sensitive to ammonia concentrations, which partlydetermines ammonia toxicity onADprocess.DuetosphericalMethanosarcinawithhigher ratioofvolume/surfacethanthatofrod-shapedMethanosaeta,

thediffusionofammoniaislessintotheMethanosarcinathan

Methanosaeta,9_{whichresultsintheirdifferentsensitivitiesto}

ammoniaconcentrations.Thus,highammoniamoreprobably causesfailureofADwhereMethanosaetaaredominatedinthe methanogensthanthatwhereMethanosarcinaaredominated. Therecoveryof ADfrom failure depends ona reconstruc-tion of methanogenic species,10 _{so that Methanosarcina}

as dominated methanogens replace Methanosaeta. This process probably causes the decrease of acetoclastic methanogenesis.11_{ThesestudiesofammoniatoxicityonAD}

process mainly focus on methanogens and methanogenic process.3,5,6,12,13

Althoughthe ammoniatoxicityonADprocesshasbeen comprehensively revealed mainly based on methanogenic microflora,3,5,12–14_{thetranscriptionalprofilesofspecific}

path-waysand genesin responsetoammonia have rarelybeen discussed.TheADprocessconsistsofADfoodweb(hydrolysis, acidogenesis,acetogenesisandmethanogenesis),sobesides methanogenesis,theother threestepsofthefoodwebalso playimportantrolesinfinalmethaneproduction.Thus,the transcriptionalprofilesofmethanogenesis-relatedprocesses are necessary to berevealed based on metatranscriptomic analysis,whichcanprovideanewsightunderlyingammonia toxicityonADprocess.

Inthisstudy,basedonmetatranscriptomicanalysis,the transcriptionalprofilesofmicrobialcommunityinresponseto lowandhighNH4+concentrations,respectively,wererevealed

inAD.Specifically,wefocusedontheexpressionsofpathways andgenesresponsibleformethaneproductionunderdifferent NH4+concentrationstofurtherrevealthemechanism

under-lyingammoniatoxicityonADprocess.

Materials

and

methods

The AD experimentof swine manure was conductedwith working volume of 2.5L digestion sludge containing 0.75L initial inoculum,and the total solid content was 7% (Sup-plementTableS1).ThehighammoniatreatmentwithNH4+

(5000mgL−1)(HN) and low ammonia treatment withNH4+

(1500mgL−1)(LN)were determinedbyaddingNH4Clatthe

beginning of AD, which was mainly based on previous report6 _{and pre-experiments. All thetreatments were}

con-ducted in triplicate at 37◦C. Seed slurry was prepared by anaerobic digestion ofswine manure (obtained from a pig farm inNeijiang, SichuanProvince,China)at37◦C,forone hydraulic retention time (HRT). After methane production reachedthefirstpeak(6thday)inthereactor,weperformed asemi-continuousfeedingmodethat500mLofdigestatewas exchangedeverythreedayswithHRTof15daysandorganic loadingrateof4.5gVS(volatilesolid)L−1day−1.The anaero-bicdigestionwasperformedfortwoHRT.Thefeedingslurry wasadjustedtothecorrespondingNH4+concentrationusing

NH4Cl.Detailsaboutparametersatthestartoffermentation

wereshowninSupplementTableS1.

Samplingandanalysis

Ateachfeeding,methanecontentinbiogas,volatilefattyacids (VFA),NH4+andpHweremeasuredtomonitorADdynamic.

Themethanecontentinbiogaswasmeasuredusingan Agi-lent6890gaschromatographysystem(AgilentTechnologies, USA),equippedwithathermalconductivitydetectorand car-riergasofargon.Theinjectionport,columnoven,anddetector wereoperatedat100,70,and150◦C,respectively.Thedaily volumeofbiogaswasdetectedbywaterreplacementmethod. TheVFAintheslurrywasdetectedusingAgilent1260Infinity liquidchromatography(AgilentTechnologies,USA),equipped withadifferentialrefractiondetector(RID)andmobilephase ofH2SO4(0.005M).Totalsolidandvolatilesolidweremeasured

basedonpreviousreport.15_NH

4+ concentrationwas

quanti-fiedwithNessler’sreagentcolorimetricmethod.16_Attheend

ofthesecondHRT,thedigestateweresampledintriplicate fortotalRNAextractionwiththeRNeasyPowerMicrobiome Kit(Cat.No.26000-50;MOBIO,USA).ThequalityofRNAwas checkedwithaNanoDrop2000spectrophotometer(Thermo, USA).RibosomalRNAwasremovedfromthetotalRNAwith theRiboMinusTMkit(Lot.No.1539791;Invitrogen,USA).The metatranscriptomic(mRNA)sequencingwasperformedusing anIlluminaHiseq2000(IlluminaInc.,USA).Thesequencing rawdatafromtotal6sampleswereuploadedtoMG-RASTwith assigned MG-RAST ID (mgs589946, mgs589949, mgs589952, mgs589955,mgs589958andmgs589961)forfurtheranalysis.17

Priortotheanalysis,thequalitycontrolpipelineinMG-RAST17

wasperformedtoremovepoorqualitysequences.The anno-tationoffunctionalprofileswasbasedontheKEGGOrthologs databaseincluding4levels.Thefunctionalcategorieswas pre-sentedaslevel2.Level3reflectedtheKEGGpathways,and level4(geneexpressionlevel)showedexpressionsofspecific

1.5

1.0

0.5

0.0

0 5 10 15 20 25 30 35 40

Fermentation time (days)

LN HN 6000 4000 2000 NH 4 + concentr ation (mg/L) 0

Daily methane production (L/L)

Fig.1–Fermentationperformanceunderhigh(HN)andlow (LN)NH4+_{concentrations.Allthedataarepresentedas} means±standarddeviations(n=3).

genes.18_{Duringannotationanalysis,thepipelineparameters}

werekeptatdefaultsettings.

Statisticalanalysis

Theammoniaeffectonthegeneralchangesofgene expres-sionswasassessedbyprincipalcoordinatesanalysis(PCoA) inR(http://www.r-project.org/),basedontheBray–Curtis dis-similarityindex,usingtheveganpackage.Thenormalityand homoscedasticityoftherawdata, andonewayanalysisof variance(ANOVA)wereperformedinSPSS21software(IBM USA).Spearman’s correlationanalysis, enrichmentanalysis andredundancyanalysis(RDA)werecalculatedinRwiththe

veganpackage.TheSpearman’spvaluewasadjustedusingthe BenjaminiandHochbergmethods.19 _{Thetaxa withaverage}

relativeabundance>0.1%andsignificantlydifferent expres-sion(p<0.05)betweenHNandLNwereselectedfornetwork analysistorevealco-responsetoammonia.Ifthecorrelation

pvaluewas<0.05,thecorrelationbetweentwotaxawas con-sideredstatisticallyrobustand shown inthenetwork. The networkwasvisualizedinCytoscapesoftware.20

Results

Anaerobicdigestionandglobalexpressionsofgenes

There were significant differences of AD performances betweenLNandHN(Fig.1andSupplementTableS1).Methane productionandcontentinthebiogaswerehigherinLNthan thatin HN(p<0.01). Theconcentrations ofaceticacid and propionicacid, and pH were higher inHN. Itshowed that highNH4+ concentrationinhibitedmethaneproductionand

resultedinsomeaccumulationsofVFA.Theglobal expres-sionsofgenesshowedobviousdifferencesbetweenHNand LN(Fig.2),whichwellcorrespondedwiththesignificant dif-ferences of performances of AD. Compared with HN, the replicatesinLNclusteredmoreclosely(Fig.2),whichimplied thathigh NH4+ probablycausedmorerandomness ofgene

expressionsinAD. 0.10 0.00 −0.10 −0.4 −0.2 0.0 0.2 0.4 PCo1 (38.3%) PCo2 (30.4%) LN HN

Fig.2–Theprincipalcoordinatesanalysis(PCoA)ofglobal expressionsofgenesinanaerobicdigestionswithhigh NH4+_{(HN)andlowNH4}+_(LN). 20 * * 15 10 5 0 Energy metabolism Amino a cid meta bolism Car boh ydr ate metabolism Translation Transcr iption Replicati on and repair Transpor t and catabolism Cell g row th and death Lipid meta bolism Environmental adaptati on Met abolism o f cof

actors and vitamins Cell m otility Membr ane tr anapor t Signal tr ansducti on Nucleotide metabolism Folding, sor ting and deg

radation LN HN Relativ e ab undance (%) *

Fig.3–Expressionprofilesoflevel2athighNH4+_(HN)and lowNH4+(LN).**significantatp<0.01;*Significantat

p<0.05.

Differentiatedexpressionsofmetabolicpathways

Inthe comparisonbetweenHNandLNbasedonpathways (level2),mostexpressionsofthesepathwayswerehigherin HN,butonlysignaltransductionwassignificantlyhigherat HN(p<0.05)(Fig.3).Surprisingly,onlyenergymetabolismwas higherinLN(p<0.01).Atlevel3,mostexpressionsofpathways were notsignificantly distinguishable between HN and LN (Table1).Thepathwayofmethanemetabolism(ko00680) (usu-ally indicating the methanogenesis in anaerobic digestion) hasahigherexpressioninLN(p<0.01),butthepathwaysof RNApolymerase(ko03020)andValine,leucineandisoleucine degradation(ko00280)hadhigherexpressionsinHN(p<0.05)

Table1–Relativeabundancesoflevel3pathwaysunderhighandlowNH4+concentrations.

Level3 LowNH4+ HighNH4+ pvalues

ABCtransporters[PATH:ko02010] 4.66±0.21 4.76±0.37 0.757

Alanine,aspartateandglutamatemetabolism[PATH:ko00250] 2.44±0.2 2.7±0.29 0.365

Aminoacyl-tRNAbiosynthesis[PATH:ko00970] 3.31±0.11 3.51±0.19 0.270

Arginineandprolinemetabolism[PATH:ko00330]a _{1.31±0.08 1.21±0.08 0.299}

Bacterialchemotaxis[PATH:ko02030] 1.11±0.12 1.26±0.2 0.405

Bacterialsecretionsystem[PATH:ko03070] 1.37±0.1 1.4±0.13 0.813

Cellcycle-caulobacter[PATH:ko04112]a _{1.45±0.11 1.64±0.01 0.069}

Citratecycle(TCAcycle)[PATH:ko00020] 1.3±0.06 1.39±0.08 0.294

Cysteineandmethioninemetabolism[PATH:ko00270] 1.16±0.18 1.15±0.07 0.955

DNAreplication[PATH:ko03030] 1.05±0.13 1.21±0.18 0.366

Flagellarassembly[PATH:ko02040] 6.65±0.86 7.88±1.69 0.414

Glycine,serineandthreoninemetabolism[PATH:ko00260] 3.27±0.21 3.36±0.26 0.716

Glycolysis/Gluconeogenesis[PATH:ko00010] 2.73±0.09 2.9±0.14 0.208

HIF-1signalingpathway[PATH:ko04066] 1.67±0.07 2.17±0.34 0.109

Histidinemetabolism[PATH:ko00340] 1.07±0.09 0.95±0.07 0.200

Methanemetabolism[PATH:ko00680]a _{10.96±0.57 6.52±0.83 0.003}

Oxidativephosphorylation[PATH:ko00190] 6.4±1.28 4.22±0.67 0.100

Pentoseandglucuronateinterconversions[PATH:ko00040] 1.04±0.06 0.97±0.12 0.483

Pentosephosphatepathway[PATH:ko00030] 1.15±0.08 1.21±0.09 0.524

Peroxisome[PATH:ko04146] 1.97±0.31 2.54±0.31 0.142

Plant-pathogeninteraction[PATH:ko04626] 1.43±0.04 1.46±0.05 0.597

Purinemetabolism[PATH:ko00230] 2.42±0.19 3.07±0.3 0.062

Pyruvatemetabolism[PATH:ko00620] 2.12±0.21 1.69±0.11 0.063

Ribosome[PATH:ko03010] 8.5±0.26 9.6±0.74 0.117

RNAdegradation[PATH:ko03018] 3.64±0.2 4.11±0.18 0.063

RNApolymerase[PATH:ko03020]b _{2.7±0.02 2.98±0.13 0.038}

Two-componentsystem[PATH:ko02020] 1.54±0.1 1.61±0.07 0.465

Valine,leucineandisoleucinedegradation[PATH:ko00280] 1.48±0.08 1.7±0.03 0.027

Taxawithaveragerelativeabundance>1%areshown.Thepvaluesrepresentthesignificancefromthecomparisonoftherelativeabundances

ofpathwaysunderhighandlowNH4+concentrations.

a _{Indicatessignificantcorrelations(p<0.05)betweentherelativeabundancesofpathwayswithdailymethaneproduction.}

b _p<0.01.

(Table 1). The relative abundances of arginineand proline metabolism(ko00330)andmethanemetabolismshowed pos-itive correlations with daily methane production (p<0.05), but those of RNA polymerase and cell cycle-caulobacter (ko04112)showednegativecorrelationswithmethane produc-tion(p<0.05)(Table1).

Inredundancyanalysis(RDA)oftherelationshipbetween theexpressionsofpathways(level3)andtheenvironmental variables (Fig. 4a),the expressionsofthepathways includ-ingmethanemetabolism,arginineandprolinemetabolism, pentosephosphatepathway(ko00030),histidinemetabolism (ko00340),oxidativephosphorylation(ko00190)andpyruvate metabolism(ko00620)positivelycorrelatedwithmethane pro-duction. The expressions of RNA degradation (ko030180), pentoseandglucuronateinterconversions(ko00040)and Cit-ratecycle(ko00020)positivelycorrelatedwithaceticacidand pH.TheexpressionsofDNA replication (ko03030)and per-oxisome(ko04146)potentially contributedtopropionicacid accumulation.

Genedifferentiatedexpressions

Besidestheoveralldifferenceofgeneexpressions(Fig.2),the enrichmentanalysiswasappliedtofurtheruncover differen-tiatedexpressionsofindividualgenesbetweenHNandLN. Probablyattributedtosignificantdifferenceoftheexpression

ofmethanemetabolism(Table1),mostofthesegeneswith significantdifferentiatedexpressions(p<0.05)wereinvolved in methane metabolism (Supplementary Table S2). These methanogenic genes mainlyencode acetyl-CoA decarbony-laseandmethyl-coenzymeMreductase.Additionally,mostof methanogenicgenesshowedsignificantpositivecorrelations withmethaneproduction(p<0.05)(SupplementaryTableS2). Besidesmethanogenicgenes,twogenesinvolvedinthe path-wayofpyruvatemetabolismshowed higherexpressionsin LN, but three and two genes involved in aminoacyl-tRNA biosynthesis(ko00970)andribosome(ko03010),respectively, showedhigherexpressionsinHN(SupplementaryTableS2). IntheRDAoftherelationshipbetweengeneexpressionsand environmental variables (Fig. 4b), it indicated that besides the methanogenic genes, the geneswhich encode glycerol kinase,phosphateacetyltransferase,transportingATPaseand pyruvateorthophosphate dikinasepositively contributed to methane production. The genes which encode ribosomal subunits,tRNAsynthetaseandphosphoribosylamineglycine ligasepositivelycontributedtopHandaceticacid.

Although there were 19 genes (each taxon with aver-age relativeabundance>0.1%) significantlydistinguishingly expressed between LN and HN (Supplementary Table S2), the co-expressions of these genes should be further veri-fied.BasedonSpearman’correlations,theco-expressionsof these genes to different NH4+ concentrations were further

1.0 1.0

Oxidative phosphorylation Pyruvate metabolism

Pentose phosphate pathway Histidine metabolism

Methane metabolism

Arginine proline metabolism

Ribosome _{Pyruvate orthophosphate dikinase}

Pentose glucuronate interconversions RNA degradation Citrata cycle DNA replication Peroxisme pH pH Acetic acid Acetic acid Propionic acid Propionic acid Methane Methane -1.0 -1.0 -1.0 -1.0 RD A2 (17%) RDA1(69%) RDA1(88%) 1.0 1.0 RD A2 (6%) Transporting ATPase Phosphate acetyltransferase Glycerol kinase Ribosomal subunits Ribosomal subunits Ribosomal subunits tRNA synthetase

Phosphoribosylamine glycine ligase

a

b

Fig.4–Redundancyanalysis(RDA)ofkeypathway(a)andgene(b)expressionswherethemethanogenicgenesmainly encodingacetyl-CoAdecarbonylaseandmethyl-coenzymeMreductasepositivelycontributetomethaneproductionbutare omittedtosimplifythefiguretobeclearer.

Oxidative phosphorylation Pyruvate metabolism Glycerolipid metabolism Methane metabolism RNA degradation Cell cycle-caulobacter Purine metabolism Aminoacyl-tRNA biosynthesis Ribosome

a

b

Fig.5–Networksofgeneexpressionsnegatively(a)and positively(b)respondingtoammonia.Thetaxawith averagerelativeabundance>0.1%andsignificant

differentiatedexpressionsbetweenLNandHNareshown.

revealed(Fig.5andSupplementaryTableS3).Theexpressions ofthe genesinvolvedin methanemetabolism,glycerolipid metabolism, pyruvate metabolism and oxidative phospho-rylation showed a co-inhibition under the high ammonia condition.Theexpressionsofmethanogenicgenesespecially thegeneswhichencodeacetyl-CoAdecarbonylasewere com-prehensively co-inhibited by high ammonia. However, the expressionsofthe genesinvolved inribosome, aminoacyl-tRNAbiosynthesis,purinemetabolism,cellcycle-caulobacter andRNAdegradationshowedco-enhancementunderthehigh ammonia condition. The expressions of more genes were

co-inhibitedunderthehighammoniacondition,which sup-portedammoniatoxicityonADprocess.

Discussion

Although the hydrolysis of substrates would cause varia-tionsofammoniaconcentrationsduringAD,inthisstudythe operations ofammonia additioninHNandLNcreated dis-tinct ammonia pressures todifferentiate ADperformances and microbialgene expression(Figs.1and 2).High ammo-niasignificantlyinhibitedADprocessbydecreasingmethane productionandcontentinthebiogas,andtheaccumulations ofVFA.Thedecreasedmethanecontentinbiogas notonly resulted inalow methaneproductionbut alsoindicateda mass of byproducts.The accumulations of VFA,especially acetic acid, implied a potential inhibition of acetoclastic methanogenesis,whichagreedwellwithpreviousreports.3,5

Although the accumulations of VFA were observedin HN, suchaccumulationsunlikelycausedexcessiveacidificationin AD,whichwasfurthersupportedbythepHvalue(7.8±0.5). Additionally,theammoniainhibitionmoreprobablyoccurred underthermophilictemperaturesthanmesophiliccondition (37◦C).3_{Thus,inthisstudy,theinhibitionofADprocesswas}

supposed to be directly attributedto ammonia toxicity on microbialactivities,ratherthanotherenvironmentalfactors regulatedbyammonia.

The microbial activities were obviously regulated by ammonia,whichwassupportedbythePCoAresult(Fig.2). In level2pathways, theexpressionsofenergymetabolism andsignaltransductionweresignificantlyinfluenced(Fig.3). The AD actually is an energy metabolism process dur-ing which energy is transferred and transformed through various metabolic pathways especially the methanogenic pathways.Theenergymetabolismmainlyincludingmethane metabolismusuallycouplestoactualmethaneproduction,1,21

so the inhibited expression of energy metabolism mainly resultedinthelowmethaneproductioninHN.Signal trans-duction is usually involved in modulating cell behaviors in response to environmental pressures such as pH and ammonia.22,23 _{Thus,ahigherexpressionofthispathwayin}

cell motility inHN coupledto that of signal transduction, whichagreedwithpreviousreport.24_{Thisfurthersupported}

microbialresponsetoammoniabybehaviorsormovements. Unexpectedly,inHNonlytheexpressionofenergymetabolism wasdepressed,butother pathwayshad higherexpressions compared to that in LN (Fig. 3). In consideration of that methanogensarenotdominantmicroorganismscomparedto bacteriasuchasFirmicutes,Bacteroidetesand Proteobacte-riainAD,25 _{it isreasonabletodeducethat thesepathways}

exceptenergymetabolismweremainlyconductedbybacteria. Thus,thehigherexpressionsofthesepathwaysinHN proba-blyindicatedhighactivitiesofbacteriaunderhighammonia condition.Thiswaslikelybecauseammonia couldimprove nutrientofnitrogenforbacterialgrowth.4_{Inlevel3,themost}

significantlydifferentiated expressionsbetweenHNandLN wasmethanemetabolism(p=0.003)whichpositively corre-latedwithmethaneproduction(Table1).Mostoftheother pathwayshadnosignificantexpressiondifferences. Interest-ingly,onlytheexpressionsofmethanemetabolism,oxidative phosphorylationandpyruvatemetabolismwerehigherinLN andpositivelycontributedtomethaneproduction(Fig.4and

Table1),buttheotherpathwayshadhigherexpressionsinHN. Thissituation wassimilarwiththatoflevel2.Thisfurther confirmedthathighammoniaprovidedsomeimprovements tosubstratemetabolismsmainlyconductedbybacteria,but inhibitedmethanogenicactivity.Consequently,althougheach stepofADfoodwebplayedcrucialrolesinactualmethane pro-duction,theammoniatoxicityonADprocessmainlytargeted methanogenicprocesstoinhibitmethaneproduction.

Ammonia inhibition on methanogenic process would resultinashiftinmethanogenicacetateutilizationfromdirect acetate cleavage toward syntrophic acetate oxidation.11,26

Thiswas mainly attributedtothe greatly decreased abun-dance of acetoclastic Methanosaeta under high ammonia condition.11 _{However, whether the expressions of genes}

involvedinacetoclasticmethanogenesiswerereducedshould berevealed.Inmethanogenicpathway,onlytheexpressions of the genes which encode acetyl-CoA decarbonylase and methyl-coenzymeMreductaseweresignificantlyhigherinLN (p<0.05),andshowedpositivecorrelationswithmethane pro-duction(p<0.05).Acetyl-CoAdecarbonylaseplaysan impor-tant role in acetoclastic pathway27 _{and methyl-coenzyme}

Mreductaseisthe keyenzymeinmethanogenesis.28_Thus,

the higher expressions of these genes encoding the two enzymes undoubtedly improved methane production. The expressionsof genes which encode acetyl-CoA decarbony-laseweresignificantlydepressedinHN,indicatingthatthe acetoclasticmethanogenesis was inhibited. Basedon tran-scriptional profile, the ammonia toxicity on acetoclastic methanogenesiswasfurtherrevealed.Additionally,ammonia inhibitedmethanogens’activitiesalsoprobablybydepressing theexpressionsofgeneswhichencodemethyl-coenzymeM reductase.Besidesmethanogenicgenes,thegenesinvolvedin pyruvatemetabolismhadsignificantlyhigherexpressionsin HN,whichprobablyimprovedtheproductionofacetyl-CoA.29 Thegenesinvolvedinaminoacyl-tRNAbiosynthesisand ribo-someweresignificantly higherexpressedinHN,indicating thatammoniapotentiallyfacilitatedtranslationprocess.13

Althoughthe expressionsofindividual key genes could generallyexplaintheammoniatoxicityonADprocess,gene

co-expressions in responseto ammonia probablyprovided a systemic sight. The inhibition of the co-expressions of the genes involved in methane metabolism, glycerolipid metabolism,pyruvatemetabolismandoxidative phosphory-lation indicatedthatthese pathwaysprobablyperformeda cooperation toregulateADefficiency thatwas mainly rep-resentedbymethaneproduction.Thecouplingrelationship betweenmethanemetabolismandoxidativephosphorylation hasbeenrevealedbefore.1 _{Theglycerolipidmetabolismand}

pyruvatemetabolismprobablyprovidedpotentialsubstrates29

formethanogenesis.Interestingly,nearlyallthegeneswhich encode acetyl-CoA decarbonylase showed inhibitionof co-expressionsunderthehighammoniacondition,whichfurther demonstratedthatacetoclasticmethanogenesiswere inhib-ited byhigh ammonia. Thisalsoshowed that these genes compared toother methanogenic geneswere unitedly and highlysensitivetoNH4+concentration.Thesegenescouldbe

usedaspotentialindicatorsforammoniatoxicityonAD pro-cess. Additionally, theininhibition ofco-expressionofthe geneswhichencodeacetyl-CoAdecarbonylaseand methyl-coenzymeMreductase,indicatingthecooperationofthetwo enzymesinmethanogenesis.

Consequently,exceptmethanogenesis,mostofpathways were not significantly influenced by high ammonia con-centration, so that they were probably notthe key factors contributingtodecreasedmethaneproduction.Thus, ammo-nia toxicity on AD process mainly targetedmethanogenic processbyinhibitingtheexpressionsofgeneswhichencode acetyl-CoAdecarbonylaseandmethyl-coenzymeMreductase tofinallydecreasemethaneproduction.Thisstudyrevealed mechanismsunderlyingmicrobialresponsetoammonia pres-sure based on gene expressions, and further discovered ammoniainhibitiononlytargetingonthegenesencodingthe abovetwoenzymes.However,theadaptionsofmicrobialgene expressionstoammonia pressure duringlongtime ADalso shouldbeconsideredinthefuturework.

Conflicts

of

interest

Authorsdeclarethattheyhavenocompetinginterests.

Acknowledgements

This work was supported by the Key Project of Educa-tion Department of Sichuan Province (No. 17ZA0215) and MajorCultivationProjectofEducationDepartmentofSichuan Province(No.17CZ0018).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.04.008.

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