Impact of process temperature and organic loading rate on cellulolytic/hydrolytic biofilm microbiomes during biomethanation of ryegrass silage revealed by genome-centered metagenomics and metatranscriptomics

RESEARCH ARTICLE Open Access Impac o p ocess empe a u e and o ganic loading a e on celluloly ic / hyd oly ic bio ilm mic obiomes du ing biome hana ion o yeg ass silage e ealed by genome-cen e ed me agenomics and me a ansc ip omics I ena Maus 1† , Michael Klocke 2† , Jaqueline De enkó 2 , Y onne S olze 1 , Michael Becks e e 3 , Ca s en Jos 2 , Daniel Wibbe g 1 , Jochen Blom 4 , Ch is ian Henke 5 , Ka ha ina Willenbüche 2 , Madis Rumming 5 , An je Rademache 2 , Al ed Pühle 1 , Alexande Sczy ba 1,5† and And eas Schlü e 1*† Abs ac Backg ound: Anae obic diges ion (AD) o p o ein- ich g ass silage was pe o med in expe imen al wo-s age wo- phase biogas eac o sys ems a low s. inc eased o ganic loading a es (OLRs) unde mesophilic (37 °C) and he mophilic (55 °C) empe a u es. To ollow he adap i e esponse o he biomass-a ached celluloly ic/hyd oly ic bio ilms a inc easing ammonium/ammonia con en s, genome-cen e ed me agenomics and ansc ip ional p o iling based on me agenome assembled genomes (MAGs) we e conduc ed. (Con inued on nex page) © The Au ho (s). 2020 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. * Co espondence: aschlue @cebi ec.uni-biele eld.de † I.M. and M.K. con ibu ed equally o his wo k. † A.Scz. and A.Schl. con ibu ed equally o his wo k. 1 Biele eld Uni e si y, Cen e o Bio echnology (CeBiTec), Genome Resea ch o Indus ial Mic oo ganisms, Uni e si ä ss . 27, 33615 Biele eld, Ge many Full lis o au ho in o ma ion is a ailable a he end o he a icle En i o nm e n a l Mi c ob i o m e Maus e al. En i onmen al Mic obiome (2020) 15:7 h ps://doi.o g/10.1186/s40793-020-00354-x (Con inued om p e ious page) Resul s: In o al, 78 bac e ial and a chaeal MAGs ep esen ing he mos abundan membe s o he communi ies, and ea u ing de ined quali y c i e ia we e selec ed and cha ac e ized in de ail. De e mina ion o MAG abundances unde he es ed condi ions by mapping o he ob ained me agenome sequence eads o he MAGs e ealed ha MAG abundance p o iles we e mainly shaped by he empe a u e bu also by he OLR. Howe e , he OLR e ec was mo e p onounced o he mesophilic sys ems as compa ed o he he mophilic ones. In con as , me a ansc ip ome mapping o MAGs subsequen ly no malized o MAG abundances showed ha unde he mophilic condi ions, MAGs espond o inc eased OLRs by shi ing hei ansc ip ional ac i i ies mainly wi hou adjus ing hei p oli e a ion a es. This is a clea di e ence compa ed o he beha io o he mic obiome unde mesophilic condi ions. He e, he esponse o inc eased OLRs in ol ed adjus ing o p oli e a ion a es and co esponding ansc ip ional ac i i ies. The analysis led o he iden i ica ion o MAGs posi i ely esponding o inc eased OLRs. The mos ou s anding MAGs in his ega d, ob iously well adap ed o highe OLRs and/o associa ed condi ions, we e assigned o he o de Clos idiales(Ace i ib io sp.) o he mesophilic bio ilm and he o de s Bac e oidales (P e o ella sp. and an unknown species), Lachnospi ales (He binix sp. and Kineo h ix sp.) and Clos idiales (Clos idium sp.) o he he mophilic bio ilm. Genome-based me abolic econs uc ion and ansc ip ional p o iling e ealed ha posi i ely esponding MAGs mainly a e in ol ed in hyd olysis o g ass silage, acidogenesis and / o ace ogenesis. Conclusions: An in eg a ed -omics app oach enabled he iden i ica ion o new AD bio ilm keys one species ea u ing ou s anding pe o mance unde s ess condi ions such as inc eased OLRs. Genome-based knowledge on he me abolic po en ial and ansc ip ional ac i i y o esponsi e mic obiome membe s will con ibu e o he de elopmen o imp o ed mic obiological AD managemen s a egies o biome hana ion o enewable biomass. Keywo ds: Me agenome assembled genomes, In eg a ed -omics, Polyomics, Anae obic diges ion, Biogas, Biocon e sion, Mic obial communi y s uc u e, Me hane, Me abolic ac i i y Backg ound Impo an pa o bioeconomical s a egies o sus ain- able and ca bon dioxide (CO 2 ) - neu al ene gy p oduc- ion is he anae obic diges ion (AD) and biome hana ion o enewable aw ma e ials. Beside manu e and slu ies om ag icul u al husband y, also ag icul u ally p oduced biomass such as maize silage (‘ene gy c ops’) o ma e ial om landscape managemen we e u ilized [1,2]. C op biomass is ich in long-chained ca bohyd a es such as cellulose, hemicellulose, and xylan and addi ionally con- ains conside able amoun s o p o eins depending on he pa icula c op. To diges c op biomass as sole subs a e and a high o ganic loading a es (OLRs), specially designed bio- gas eac o s a e ad an ageous such as s aged deg ada ion s ep (‘phase’) sepa a ed eac o sys ems consis ing o a cellu- lolysis / hyd olysis e men e and a downs eam me hano- genesis eac o . Compa ed o single phase eac o s, hese wo-s age wo-phase eac o s possess se e al ad an ages, o example, he mo e s able ope a ion (unde pa icula p ocess condi ions) combined wi h highe bioene gy yields [2–6]. The deg ada ion o high-molecula compounds e- qui es he di ec access o mic obial specialis s. Acco d- ingly, c op biomass is colonized by a celluloly ic bio ilm which composi ion a ies wi h he abio ic en i onmen al condi ions, as example, he p ocess empe a u e, bu also elies on he physico-chemical cha ac e is ics o c op ma e ial and p ocess liquids [7]. Once he bio ilm is es ablished, i unc ions as a coope a i e conso ium leading o enhanced biomass deg ada ion and, in conse- quence, o biogas p oduc ion [8,9]. Comp ehensi e mic obiome analyses by means o me a- genome sequencing e ealed ha many biogas mic o- biome membe s could no be classi ied down o he species le el and hence a e cu en ly unknown (‘mic obial da k ma e ’)[10,11]. As example published p e iously, in he me agenome da ase de e mined o a biogas plan op- e a ed unde he mophilic empe a u e egime, only 18% o he included 16S RNA gene sequences we e assignable o a axonomically es ablished genus [10]. In addi ion, in- o ma ion on he me abolic ac i i y o biogas bio ilm mi- c obial communi ies is only udimen a ily a ailable. Mos o he co esponding s udies ocus on he me hanogenic sub-communi y, while he me abolic po en ial o he en- i e biogas bio ilm mic obiome emains poo ly cha ac e - ized [12,13]. Fo his p opose, he in eg a ion o di e en -omics app oaches (in eg a ed o poly-omics), as example, he combina ion o me agenome wi h me a ansc ip ome da ase s, is indispensable o dis inguish be ween me abol- ically ac i e and less ac i e mic obial species. Genome- cen e ed biogas mic obiome analyses al eady disclosed and p ospec i ely will disclose u he unc ionali ies and in e ac ions o keys one mic obiome membe s [14–21]. The deg ada ion o plan biomass by hyd oly ic enzymes o su ace a ached celluloly ic and hyd oly ic bac e ia and he subsequen seconda y e men a ion s ill ep esen bo - lenecks in he enginee ed op imiza ion o biogas p ocesses. Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 2 o 21 Fo economically op imal AD p ocess ope a ion, celluloly ic / hyd oly ic bio ilms adap ed o maximal OLRs and, wi h special espec o he e men a ion o subs a es wi h high con en s o ni ogen-con aining compounds such as p o- eins and pep ides (e.g., g ass silage), mic obiomes ea u ing ole ance o high ammonium / ammonia con en s a e es- sen ially equi ed. I was hypo hesized ha such bio ilm membe s, in pa icula unc ional keys one species, will become ecognizable by hei inc easing abundances and ansc ip ional ac i i ies unde demanding p ocess condi ions. To un a el he s uc u e, unc ionali y, and me abolic ac- i i y o such celluloly ic / hyd oly ic bio ilm mic obiomes, in his s udy, an in eg a ed -omics app oach was applied consis ing o pa allel mic obial me agenome and me a an- sc ip ome analyses. To ob ain di ec access o hese mic o- biomes, bio ilm samples g own on he su ace o yeg ass silage diges ed in he hyd olysis eac o s (HR) o wo-s age wo-phase biogas eac o sys ems (Fig. 1)ope a edunde mesophilic (37 °C) and he mophilic (55 °C) empe a u e e- gime and a wo (low and inc eased) OLRs we e analyzed. Me abolic econs uc ion o candida e me agenome- assembled genomes (MAGs) and co esponding genome- cen e ed ansc ip ome analyses p o ided insigh s in o li e- s yles and ac i i ies o adap ed species. Resul s Biogas eac o pe o mance and long- e m mic obial dynamics Fou biogas eac o sys ems we e ope a ed in pa allel o e a pe iod o longe han 750 days esul ing in wo (biological) eplica es o mesophilic (M1, M2) and wo eplica es o he mophilic condi ions (T1, T2) (Fig. 1). Sampling o subs a e su ace a ached bio ilms was con- duc ed o wo di e en OLRs, i.e., 500 g (low OLR) and 1500 g (inc eased OLR) o pe ennial yeg ass silage. The summa ized a e age biogas yields om hyd oly ic eac o s (HR) and anae obic il e s (AF) we e cons an ly 601 ± 18 l no malized o s anda d empe a u e and p essu e (L N ) pe kg ola ile subs ances (VS) (M1) and 599 ± 26 L N kg VS −1 (M2) o he eac o sys ems ope - a ed unde mesophilic empe a u e egime wi h an a e - age me hane con en o 56 ± 2% ( / ) (Fig. 2). Unde he he mophilic empe a u e egime, he summa ized sys- ems’biogas yields we e sligh ly highe wi h a e age alues o 645 ± 27 L N kg VS −1 (T1) and 644 ± 19 L N kg VS −1 (T2) wi h a sligh ly lowe a e age me hane con- en o 54 ± 2% ( / ) each. The a e age me hane yields anged om 337 ± 20 L N kg VS −1 (M2) o 348 ± 19 L N kg VS −1 (T2) which is less han 5% lowe han he e e - ence alue o , on a e age, 353 L N kg VS −1 as de e mined by s anda d ba ch e men a ion es s acco ding o he Ge man echnical s anda d VDI 4630. E en i , in gen- e al, he me hane con en in he biogas was highe in he AF han in he HR (mesophilic, 71 ± 3% s. 51 ± 2%; he mophilic, 70 ± 2% s. 50 ± 2%), mos o he biogas and me hane was p oduced in he HR (mesophilic, 74 ± 6% and 67 ± 6% on a e age; he mophilic, 79 ± 4% o 81 ± 4% and 73 ± 4% o 76 ± 4% depending on he e- spec i e p ocess s a us). The main p oduc o bac e ial e men a ion in he HR was ace ic acid. Highes concen a ions we e de e mined o e men a ion day 2 wi h alues anging om 0.80 g L −1 Fig. 1 Flow scheme (a) and pic u e (b) o he wo-s age wo-phase biogas eac o sys em Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 3 o 21 (s a -up phase) o 5.30 g L −1 (OLR 1500 g silage) and unde he he mophilic condi ions, om 0.91 g L −1 (s a - up phase) o 4.21 g L −1 (OLR 1500 g silage). P opionic acid was p oduced only in mino amoun s o up o 0.58 g L −1 (mesophilic) and 0.43 g L −1 ( he mophilic). The ola ile a y acids (VFA) accumula ion h oughou he ongoing e - men a ion p ocess was no obse ed. Due o he ni ogen con en o he silage o 7.6 g kg FM −1 ,a NH 4+ accumula ion o up o 2.3 g L −1 (mesophilic) and 2.7 g L −1 ( he mophilic) was obse ed in he p ocess luids du ing he e men a ion and wi hin he en i e biogas eac o sys em (Fig. 2). In con as o he e men a ions a mesophilic em- pe a u es, unde he mophilic empe a u e egime also an accumula ion o cy o oxic NH 3 o up o 1.3 g L −1 occu ed. Fig. 2 Biogas and me hane yields, and NH 4+ - and NH 3 -con en s in he biogas eac o e luen s. Bac e ial 16S RNA gene a ge ing TRFLP analyses esul s a e shown exempla y o di e en ime poin s a di e en p ocess condi ions. Do ed lines indica e ime poin s o sampling o NGS. ▲,△, hyd olysis eac o (HR) o eac o sys em M1 (mesophil) esp. T1 ( he mophil);▼,▽, downs eam AF o eac o sys em M1 (mesophil) esp. T1 ( he mophil); ■,□, HR o eac o sys em M2 (mesophil) esp. T2 ( he mophil); ◆,◇, AF o eac o sys em M2 (mesophil) esp. T2 ( he mophil); L N , li e s no malized o 0 °C and 1013 hPa; VS, ola ile subs ances; OLR, o ganic loading a e Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 4 o 21 As e ealed by bac e ial 16S RNA gene a ge ing TRFLP analysis, he bac e ial communi y s uc u e had adap ed du ing he ongoing biome hana ion expe imen (Fig. 2). I is assumed ha adap a ion o he communi y p ima ily occu ed as esponse o he inc ease in OLR. Likewise, also he inc ease in NH 4+ concen a ions and, in pa icula du ing he he mophilic e men a ions, he inc ease in NH 3 concen a ions may had a ec ed he communi y composi ion. Celluloly ic / hyd oly ic bio ilm communi y s uc u e To cha ac e ize he s uc u e o he bac e ial bio ilms es ablished on he su ace o yeg ass silage diges a e in he mesophilic and he mophilic HRs, nex gene a ion sequencing (NGS) o he 16S RNA gene was pe o med. Due o he used p ime se s, p ima ily 16S RNA genes o he domain Bac e ia we e de ec ed. 16S RNA gene sequences o me hanogenic A chaea we e only de ec ed o mesophilic bio ilm samples (in maximum, wi h an abundance o 1.2% in case o he HR bio ilm sample M1 OLR1500 ) and assigned o genus Me hanosae a, i.e., Me hano h ix. O e all, 66% o he 16S RNA gene sequences we e classi ied in o ope a ional axonomic uni s (OTUs) ha could be assigned o known gene a. 34% emained un- assigned a genus ank (Addi ional ile 1). In gene al, he he mophilic bio ilms exhibi ed a sligh ly lowe di e si y han he mesophilic bio ilms (Shannon indices in a e - age 2.9 s. 3.2). In hemesophilicbio ilms,anumbe o gene awe e exclusi ely de ec ed belonging o he classes Clos- idia (i.e., Cellulosily icum,Lachnospi a,Anae ospo o- bac e ,Bu y i ib io,andEpulopiscium), Bac e oidia (i.e., Bac e oides,andPe imonas), Tissie ellia (i.e., Sedimen ibac e )(Fig.3, Addi ional ile 2). In gene al, he abundance o pa icula OTUs a ied be ween he biological eplica es and OLRs indica ing a ying mic obiome s uc u e. Fig. 3 Taxonomic p o iling o he hyd olysis eac o (HR) bio ilm mic obiome. Rela i e abundances a e shown o he mos abundan gene a o mic obial sub-communi ies as de e mined by 16S RNA gene amplicon NGS. M1, M2, mesophilic eplica es; T1, T2, he mophilic eplica es; OLR500, OLR1500, o ganic loading a es o 500 o 1500 g yeg ass silage. Fo u he de ails on sample denomina ion, e e o Fig. 2 Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 5 o 21 In he he mophilic bio ilms, membe s o he gene a Ruminiclos idium and Halocella (phylum Fi micu es, class Clos idia), and Tepidimic obium (class Tissie ellia) we e ound, which we e no o only de ec ed in mino amoun s in he mesophilic bio ilms. Also in hese bio- ilms, he abundance o OTUs a ied be ween biological eplica es and OLRs. Some gene a we e p esen in all bio ilms, such as Ruminococcus,De lu ii alea, and Mobi- li alea (class Clos idia), bu all showing subs an ial di e ences in abundance in mesophilic and he mophilic bio ilms (Fig. 3, Addi ional ile 2). Unde mesophilic empe a u e egime, he inc ease in OLR had a posi i e e ec on he abundance o some membe s, e.g., o he gene a P o einiphilum (OLR 500 g: 0.7 esp. 0.4% s. OLR 1500 g: 3.7 esp. 1.3%) and De lu- ii alea (OLR 500 g: 0.1 esp. 0.9% s. OLR 1500 g: 1.8 esp. 4.8%). O he bio ilm membe s dec eased in abun- dance a e inc ease o OLR, such as membe s o gene a Anae ospo obac e (OLR 500 g: 5.0 esp. 6.0% s. OLR 1500 g: 0.8 esp. 0.9%) and Cellulosily icum (OLR 500 g: 15.2 esp. 12.4% s. OLR 1500 g: 7.8 esp. 5.7%) (Add- i ional ile 2). A he mophilic empe a u es, only he genus Mobili- alea bene i ed om he inc eased OLR (OLR 500 g: 8.8 esp. 9.6% s. OLR 1500 g: 17.2 esp. 11.9%) (Addi ional ile 2). All o he gene a emained a mo e o less simila abundances. Thus, i can be assumed ha Mobili alea species a e mo e ole an owa ds inc eased NH 3 con- cen a ions compa ed o o he species. MAG abundance in he celluloly ic/hyd oly ic bio ilms depending on he empe a u e and he OLR To de e mine and cha ac e ize di e en ial abundances o species ep esen ed by me agenomically assembled genomes (MAGs) in he celluloly ic/hyd oly ic bio ilms es ablished on he su ace o yeg ass silage, mic obial me agenome sequencing ollowed by a combined assem- bly o sequence da a and genome binning we e applied (Addi ional ile 3). In o al, 157 MAGs we e compiled and axonomically classi ied (Addi ional ile 4). 74 MAGs we e assigned o he domain Bac e ia, i e MAGs o A chaea, and 78 emained unclassi ied a domain le el. A he phylum le el, he MAGs we e alloca ed o he Fi micu es (55), Bac e oido a (12), Eu ya chaeo a (5), Spi ochae o a (2), Fib obac e o a (1), and Cloacimo- nado a (1). A he amily le el, only 53 MAGs we e clas- si iable among o he s o he Lachnospi aceae (19), Bac e oidaceae (4), Ruminoclos idiaceae (3), Clos idia- ceae (3), indica ing occu ence o so a unknown mic obial species and/o insu icien ep esen a ion o adequa e e e ence genomes in da abases ha a e a ail- able o compa a i e analyses. To unco e he ela i e abundances o he compiled MAGs in he HRs analyzed, me agenome sequences ob ained om he mesophilic and he mophilic mic o- bial communi ies we e mapped on o he MAG se- quences. Only 78 MAGs ea u ing con amina ion a es below 10% we e conside ed o his app oach. Deepe me agenome sequencing would ce ainly ha e imp o ed he comple eness and numbe o compiled MAGs. How- e e , sequencing dep h always is a ade-o be ween in- cu ed cos s and expec ed esul s. P incipal componen (PCA) analysis o MAG abun- dance p o iles e ealed close clus e ing o eplica es con- i ming ep oducibili y o he ea men s ca ied ou (Fig. 4a). Mo eo e , empe a u es (mesophilic s. he mophilic) led o a clea sepa a ion o MAG abun- dance p o iles in he PC plo s. Likewise, he OLRs (OLR500 s. OLR1500) di e en ia e MAG abundances. Howe e , his e ec is a mo e p onounced o he mesophilic sys ems as compa ed o he he mophilic ones (Fig. 4a). MAG abundance p o iles a e isualized o all condi- ions es ed ( wo empe a u es and wo OLRs in epli- ca es) in hea -maps o he 78 MAGs es ed (Fig. 4b). Clus e analysis e ealed ha he empe a u e is he mos impo an ac o ha d i es shaping o he com- muni y ollowed by he OLR. Replica es a e e y simila o each o he ega ding MAG abundance p o iles unde he condi ions es ed. Rela i e abundances o he ollowing MAGs signi i- can ly inc eased (log 2 old-change o > 2 and –log 10 (p- alue) o > 0.05) unde mesophilic condi ions when he OLR was aised o 1500 g: MAG 13, 40, 48, 84, 85, 107, 109, 111, 132, 145 and 146 as shown by olcano plo analysis (Fig. 4c). Rela i e abundances o se e al mo e MAGs dec eased unde high OLRs. Unde he mophilic condi ions, he MAGs 50, 68, 106 and 146 inc eased in abundance upon OLR aise (Fig. 4d). Responsi e MAGs ea u ing comple eness alue o mo e han 50% and con- amina ion less han 10% we e u he analyzed ega ding hei gene ic po en ial and ansc ip ional ac i i y. These a e MAGs 40, 50, 85, 107, 109, 111 and 145 (see below). Func ional po en ial o MAGs posi i ely esponding o inc eased OLR To gain insigh s in o he unc ional po en ial o MAGs posi i ely esponding o inc eased OLRs, gene ic de e - minan s o u iliza ion o ca bohyd a es (Fig. 5) as well as he key enzymes o AD pa hways we e analyzed (Add- i ional ile 5). The gene ic de e minan s we e ca ego ized acco ding o he ou s ages o he AD p ocess, namely hyd olysis, acidogenesis, ace ogenesis and me hanogen- esis as desc ibed p e iously [23]. Rega ding he unc ional con ex ‘hyd olysis’, he gene ic po en ial o u iliza ion o ca bohyd a es was cha ac e ized in he selec ed bac e ial MAGs. Genes en- coding ca bohyd a e-ac i e enzymes we e de e mined by Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 6 o 21 applying he HMM-based Ca bohyd a e-ac i e enzyme anno a ion da abase dbCAN 7 [24] (Fig. 5). Be ween 45 and 236 genes encoding cellulosomal p o eins o en- zymes wi h p edic ed ac i i ies on ca bohyd a es we e iden i ied in each o he bac e ial MAGs analyzed. Ob ained esul s subdi ided he analyzed MAGs in o wo g oups. G oup I membe s we e p edic ed o en- code cellulosome s uc u es equi ed o e icien deg- ada ion o cellulose, comp ising docke in-con aining glycosyl hyd olases (GHs), co esponding cohesin- con aining sca oldins, and enzymes ac ing on la ge ca bohyd a e molecules. Some o he iden i ied en- zymes con ain ca bohyd a e-binding mo i s. MAG 50 ( amily De lu ii aleaceae), MAG 85 (phylum Fi mi- cu es) and 145 (a ilia ed o he genus Jeo galibaca) ep- esen g oup I membe s. In gene al, o celluloly ic/ hyd oly ic bio ilms, he MAGs belonging o g oup I a e o g ea impo ance, since hey ep esen bac e ial can- dida es ea u ing he po en ial o e icien decompos- i ion o complex ca bohyd a es such as cellulose, hemicellulose and xylan. The emaining MAGs we e classi ied o g oup II ep e- sen ing seconda y e men a i e bac e ia mainly u ilizing mono-, di- and oligosaccha ides o ene gy p oduc ion as supplied by g oup I bac e ia. G oup II comp ises MAGs lacking genes o p o eins in ol ed in cellulosome Fig. 4 Al e a ion o abundance o 157 me agenome-assembled genomes (MAGs) de ec ed in HR bio ilms a mesophilic (M) and he mophilic (T) p ocess empe a u e in esponse o he inc ease o o ganic loading a e (OLR) om 500 o 1500 g yeg ass silage as deduced om me agenome da a. PCA plo s a e shown o p inciple componen analyses o scaled and cen e ed log ans o med me agenome ead coun s mapped on MAGs (a). The hie a chical clus e ing o log ans o med abundance alues o 78 (con amina ion below 10%) selec ed MAGs de ec ed in HR bio ilms is isualized in (b). G een, mesophilic HR bio ilms; blue, he mophilic HR bio ilms. Fo u he de ails on sample denomina ion, e e o Figs. 2and 3. Al e a ion in abundance alues o 78 MAGs selec ed in esponse o he inc ease o OLR om 500 o 1500 g yeg ass silage a mesophilic (c) and he mophilic (d) p ocess empe a u e. Colo s o he MAGs indica e axonomic a ilia ion (g een, Fi micu es; blue, Bac e oido a; iole , A chaea; pink, Cloacimonado a; ligh g een, Fib obac e o a; da k ed, Spi ochae o a; ed, unknown a ilia ion). * indica e MAGs wi h comple eness abo e 50% and con amina ion a e less han 10% as lis ed in Table 1and in Addi ional ile 4 Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 7 o 21 o ma ion and assembly. Howe e , due o he incomple e- ness o hese MAGs (Table 1), i canno be excluded ha cellulosome genes we e missed du ing he assembly and binning p ocesses. Fu he mo e, gene ic de e minan s encoding key en- zymes equi ed o u iliza ion o di e en o ganic mole- cules such as py u a e, lac a e, e hanol, ace a e, p opiona e and bu y a e ep esen ing impo an me abo- li es o he acidogenesis and ace ogenesis we e analyzed in he MAGs selec ed (Addi ional ile 5). As examples, he bac e ial MAGs 109 (P o einiphilum sp.) and 145 (Jeo galibaca sp.) encode high numbe s o key genes ea u ing p edic ed unc ions in py u a e me abolism (be ween 14 and 21), also ep esen ing he KEGG (map 00620) modules o u iliza ion o lac a e (be ween 5 and 7) and ace a e (8) only in case o he MAG 145. In MAG 145, essen ial genes encoding enzymes o he Wood- Ljungdahl pa hway (8) we e iden i ied, which plays an im- po an ole in ca bon ixa ion and ace a e u iliza ion. Du ing ace ogenesis, se e al bac e ial species u ilize p opionic acid employing he me hylmalonyl-CoA o he ac ylyl-CoA pa hway o he p opanoa e me abolism. The bac e ial MAGs 109 (P o einiphilum sp.) and 145 (Jeo ga- libaca sp.) possess se e al genes (be ween 5 and 9) o enzymes ha we e assigned o he me hylmalonyl-CoA b anch o he p opanoa e me abolism. Fu he mo e, MAGs 109 and 145 addi ionally encode he me hylmalonyl-CoA mu ase Mu (EC 5.4.99.2) and he me hylmalonyl-CoA/e hylmalonyl-CoA epime ase (EC 5.1.99.1) ep esen ing he key enzymes o his me abol- ism as desc ibed by Siko e e al. [23]. The e o e, hese MAGs we e p edic ed o be in ol ed in he p opionic acid me abolism in he analyzed celluloly ic/hyd oly ic bio ilms. Mo eo e , MAGs 50, 85, 109 and 145 mos p obably a e in ol ed in bu anoa e me abolism since hey possess be ween 2 and 6 o key genes classi ied o he bu anoa e pa hway (KEGG map00650). The bu a- noa e pa hway is no comple ely encoded in he MAGs analyzed. Howe e , genes encoding ele an key en- zymes desc ibed by Siko a e al. [23] we e iden i ied in he genomes o hese Bac e ia. The MAG 109 ha bo s a gene encoding PFL-py u a e o ma e lyase (EC 2.3.1.54), whe eas MAG 145 possesses he gene encoding bu y a e kinase (EC 2.7.2.7) indica ing he impo ance o hese bac e ia o AD a mesophilic empe a u es. The o ma ion o me hane, he las s ep o AD, is pe - o med by membe s o he phylum Eu ya chaeo a.In he analyzed bio ilms, MAG 111 (genus Me hanobac e - ium sp.) was de ec ed as mos abundan a chaeon posi- i ely esponding o he inc ease in OLRs. Twen y- wo genes encoding key enzymes o he hyd ogeno ophic me hanogenesis pa hway we e iden i ied in his MAG illus a ing he impo ance o his pa hway unde inc eased OLRs. T ansc ip ional ac i i y o MAGs in esponse o empe a u e and OLR To de e mine he ansc ip ional ac i i ies o he com- piled MAGs, he me a ansc ip omes om he di e en eac o sys ems we e sequenced cap u ing he p e ailing condi ions ( empe a u e: mesophilic s. he mophilic, OLRs o 500 g s. 1500 g). Mapping o he ansc ip ome sequences o each da ase o he assembled con igs allowed de e mina ion o he MAG’s ansc ip ional ac- i i ies unde he condi ions es ed. P incipal componen (PCA) analysis e ealed clea sepa a ion o he MAG’s ansc ip ional ac i i y pa e ns in ela ion o he empe a u e (mesophilic and he mo- philic) and he OLRs 500 g and 1500 g (Fig. 6a). How- e e , compa ed o he co esponding analysis based on Fig. 5 Genes encoding ca bohyd a e-ac i e enzymes p edic ed o bac e ial me agenome-assembled genomes (MAGs) mos abundan in hyd olysis eac o (HR) bio ilms. * These MAGs showed inc eased ansc ip ional ac i i y in esponse o he o ganic loading a e and we e he e o e addi ionally analyzed Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 8 o 21 Table 1 Taxonomic a ilia ions and cha ac e is ics o selec ed me agenome-assembled genomes (MAGs) mos abundan in HR bio ilms MAG Taxonomic a ilia ion 1 MAG ea u es Phylum Class O de Family Genus Comle eness 2 [%] Con amina ion 2 [%] Size [bp] GC con en [%] No. o genes No. o n genes No. o RNA genes Bac e ia 13 Fi micu es Unknown Unknown Unknown Unknown 29.74 3.43 1,239,774 44.81 1159 3 31 17* Bac e oide es Bac e oidia Bac e oidales Bac e oidaceae P e o ella sp. 68.02 8.98 2,689,597 48.90 2294 2 39 18* Fi micu es Clos idia Lachnospi ales Lachnospi aceae Ace i ib io sp. 68.50 3.65 2,855,089 39.03 2917 n/d 3 15 40* Fi micu es Clos idia Lachnospi ales Lachnospi aceae He binix sp. 68.42 7.66 1,849,940 36.65 1755 n/d 8 48 Fi micu es Clos idia Lachnospi ales Lachnospi aceae He binix sp. 26.48 2.63 770,644 37.4 614 n/d 3 50 Fi micu es Clos idia Lachnospi ales De lu ii aleaceae Unknown 51.74 6.14 1,725,349 31.02 1775 n/d 31 68 Fi micu es Clos idia Lachnospi ales Lachnospi aceae He binix sp. 52.17 4.77 2,358,488 46.61 2310 2 27 84 Fi micu es Clos idia Lachnospi ales Unknown Unknown 35.22 5.14 1,970,476 38.81 1672 2 17 85 Fi micu es Unknown Unknown Unknown Unknown 80.11 3.08 2,011,311 47.98 2076 n/d 33 106 Fi micu es Clos idia Clos idiales Clos idiaceae Clos idium sp. 33.09 4.39 883,787 31.79 812 n/d 17 107* Fi micu es Clos idia Clos idiales Clos idiaceae Clos idium sp. 50.06 6.97 1,539,556 29.37 1648 n/d 17 108* Bac e oide es Bac e oidia Bac e oidales Unknown Unknown 79.01 8.21 2,032,215 47.60 2025 1 29 109 Bac e oide es Bac e oidia Bac e oidales Dysgonomonadaceae P o einiphilum sp. 63.52 7.19 2196,928 45.07 2196 1 27 132 Fi micu es Clos idia Unknown Unknown Unknown 26.26 2.37 1,284,977 37.03 1286 n/d 16 134* Fi micu es Clos idia Lachnospi ales Lachnospi aceae Kineo h ix sp. 78.46 9.27 2,788,291 40.86 2946 n/d 31 145 Fi micu es Bacilli Lac obacillales Ae ococcaceae Jeo galibaca sp. 63.37 9.48 2,346,037 46.04 2647 n/d 14 146 Fi micu es Bacilli Unknown Unknown Unknown 33.22 0.00 861,844 38.49 705 1 13 A chaea 111 Eu ya chaeo a Me hanobac e ia Me hanobac e iales Me hanobac e iaceae Me hanobac e ium sp. 73.74 8.57 2,134,563 37.98 2406 2 51 1 GTDB-Tk based classi ica ion 2 Fo de ails, e e o [22] and Addi ional ile 4 3 n/d = no de ec ed * MAGs which showed inc eased ansc ip ional ac i i y in esponce o he o ganic loading a e (OLR) Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 9 o 21 Table 2 Mos ac i ely ansc ibed genes o enzymes in ol ed in ca bohyd a e o p o ein u iliza ion o selec ed HR bio ilm MAGs (Con inued) Me agenome assembled genome (MAG) 1 Assumed axonomic a ilia ion Analyzed HR 2 bio ilm sample Gene ansc ip s in ol ed in ca bohyd a e o p o ein u iliza ion P edic ed unc ional ole in AD 4 GenID Gene leng h Pu a i e gene p oduc Gene EC numbe T ansc ip s mapped No malized numbe o ansc ip s [TPM] 3 Bin_108_01184 441 50S ibosomal p o ein L15 plO n/s 14 0.2 134 Lachnospi ales (Kineo h ix sp.) T2 OLR1500 Bin_134_02838 177 T ansi ion s a e egula o y p o ein ab B n/s 1527 181 Hyd olysis Bin_134_00038 555 Hypo he ical p o ein n/s n/s 491 7 Bin_134_00185 291 ATP-dependen Clp p o ease, p o eoly ic subuni clpP n/s 81 3 Bin_134_00184 1278 ATP-dependen Clp p o ease, ATP-binding subuni clpX n/s 622 3 Bin_134_00305 906 HTH- ype ansc ip ional egula o yo A n/s 324 2 Bin_134_02093 1110 T ehalose impo ATP-binding p o ein acm 3.2.1.17 234 1 Bin_134_00012 570 Amylopullulanase pulA 3.2.1.41 74 1 Bin_134_00958 3558 Py u a e- la odoxin oxido educ ase ydbK 1.2.7.1 623 1 1 The i s i e ansc ip s o each MAG ep esen he op ansc ip s o he co esponding MAG 2 Hyd olysis eac o 3 T ansc ip s pe million a e aged o da ase s om wo echnical eplica es 4 Anae obic diges ion n/s No speci ied Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 16 o 21 p ocess de ails we e as p e iously published by [3]. S a -up o all e men e s we e pe o med using liquid e men e ma e ial om a biogas plan con e ing ca le manu e in co-diges ion wi h g ass and maize silage and o he biomass a a ying concen a ions and a mesophi- lic empe a u es. Silage o pe ennial yeg ass (Lolium pe enne L.) was diges ed as sole subs a e in ba ches o a ying amoun s wi h e en ion imes o 28 d (s o age o bale silage a −20 °C, cu ing leng h 3 cm), ola ile subs ances (VS) 32% o esh mass (FM), o al Kjeldahl ni ogen 7.6 g kg FM −1 ,NH 4+ -N 0.7 g kg FM −1 , ace ic acid 2.6 g kg FM −1 , p opionic acid < 0.04 g kg FM −1 , lac ic acid 2.6 g kg FM −1 , e hanol 2.2 g kg FM −1 , C/N a io 19.3, chemical oxygen demand (COD) 357.7 g kg FM −1 , analysis o chemical p ope ies acco ding o [6]. The o al Kjeldahl ni ogen is an app oxima ion o a p o ein con en in he sample. The a e age pH had a alue be ween 7 (a he beginning o e e y expe imen ) and 8 (a he end o e e y expe i- men a e 28 days). No spoilage was obse ed in he sil- age. Biogas yields we e calcula ed as li e s no malized o 0 °C and 1013 hPa (L N ) pe kilog am ola ile subs ances (kg VS ). Fo chemical analysis, samples we e aken om he e luen s o HR and AF. Fo sequencing o 16S RNA gene amplicon lib a ies, mic obial me agenomes, and mic obial me a ansc ip- omes, samples we e aken om he silage diges a e in he HR diges ed o 2 d. A his ime poin , high AD a es we e de ec ed as indica ed by he as inc ease o ola ile a y acids (VFA), e.g., ace ic acid. Sampling was pe o med a wo di e en o ganic loading a es (OLRs), i.e.,ba ch- e men a ion o 500 g (denomina ed as “low OLR”, samples M OLR500 and T OLR500 ) and 1500 g silage (denomina ed as “inc eased OLR”, samples M OLR1500 and T OLR1500 ) (Fig. 2). Excess ai was emo ed and he bo le wi h app oxima ely 200 mg o silage diges a e was igh ly closed wi h a sc ew cap. The bio ilm on plan ma e ial su aces o igina ing om he diges a e sample was de ached using a s e ile scalpel in an anae obic chambe . Fu he de ails we e desc ibed by [12]. All samples we e s o ed a −20 °C un il u he analysis ex- cep samples o RNA isola ion, which we e p ocessed immedia ely a e sampling. Ex ac ion o o al mic obial genomic DNA To al mic obial communi y DNA was ex ac ed om su - ace a ached bio ilms by using he Fas DNA™Spin Ki o Soil (MP Biomedicals, USA) wi h Lysing Ma ix E Tubes acco ding o he manu ac u e ’s ins uc ions. Mechanical cell dis up ion was pe o med using he Fas P ep®-24 In- s umen (MP Biomedicals, USA) o wo imes a 6500 pm (speed 5) o 20 s. Fu he , wo washing s eps using SEWS-M we e accomplished. Finally, he DNA was elu ed in 100 μl DES. Two independen echnical eplica es o each HR we e p epa ed, and subsequen ly pooled in equi- mola amoun s oge he o collec su icien DNA ma e ial o sequencing pu poses (Addi ional ile 7). Quali y and quan i y o ex ac ed DNA we e e alua ed by gel elec o- pho esis and pho ome ic analysis (NanoPho ome e , Implen). All DNA samples we e s o ed a −20 °C un il u he p ocessing. Te minal es ic ion agmen leng h polymo phism (TRFLP) inge p in ing The mic obial communi y dynamics du ing he ope - a ion o he biogas eac o sys ems we e moni o ed by DNA-based TRFLP analysis a ge ing he bac e ial 16S RNA gene acco ding o he p o ocol p e iously pub- lished by [12] wi h he modi ica ions as published by [45]. TRFLP p o iles we e de e mined in iplica es o mic obial DNA samples pu i ied om he HR e luen a e 28 d ba ch e men a ion. TRFLP inge p in p o- cessing and subsequen analysis we e pe o med acco d- ing o [46] using BioNume ics 7.1 so wa e (Applied Ma hs, Belgium). Simila i ies o inge p in p o iles we e calcula ed using Pea son co ela ion wi h 0.5% op imiza ion, clus e analysis was pe o med applying he unweigh ed pai g oup me hod wi h a i hme ic mean (UPGMA) algo i hm. Nex -gene a ion-sequencing (NGS) o 16S RNA gene amplicon lib a ies The mic obial communi y s uc u es we e axonomically cha ac e ized by high- h oughpu nex gene a ion sequen- cing (NGS) o 16S RNA gene amplicon lib a ies as de- sc ibed p e iously [47]. The lib a ies we e cons uc ed using he p ime s 515F (5′- CTACGGGNGGCWGCAG - 3′) and 806R (5′- GACTACHVGGGTATCTAATCC - 3′) ampli ying he V3 and V4 egions o he bac e ial 16S RNA gene [48]. Two biological eplica es pe sample we e analyzed (Addi ional ile 7). Ob ained sequence eads we e used o i e a i e ead pai me ging applying he FLASH so wa e [49]. Subsequen ly, he QIIME NGS analysis pipe- line was applied o amplicon da a p ocessing as desc ibed p e iously [29]. Ob ained OTUs we e clus e ed a he 97% sequence iden i y le el applying he QIIME NGS analysis pipeline. Mic obial me agenome lib a y p epa a ion, NGS, and assembly o genomes om me agenome da ase s Fo lib a y p epa a ion, mic obial DNA samples we e pu i- ied using he Genomic DNA Clean & Concen a o Ki (Zymo Resea ch, USA). Fo each sample, DNA om wo subsamples (i.e., echnical eplica es) we e ex ac ed. Fo se- quencing, 1 μg o o al DNA was shea ed o app oxima ely 430 bp agmen s using a ocused-ul asonica o (Co a is M220,Wobu n,MA,USA).Finally, he Illumina T uSeq® Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 17 o 21 DNA PCR- ee sample p epa a ion ki (Illumina, Eindho- en, Ne he lands) was used o cons uc he sequencing li- b a ies, which we e sequenced on he Illumina HiSeq 1500 sequence using he Illumina HiSeq Rapid SBS Ki 2 (Illu- mina, Eindho en, Ne he lands), ollowing a 2 × 250 indexed high ou pu un p o ocol. Fu he mo e, Megahi ool ( 1.0.2) [50](command line se ings: --p ese s me a --min-con ig-len 1000) was used o assembly o he pooled sequencing da a o all samples applying a k-me sizes o 21, 41, 61, 81 and 99 (i e a i e assembly). Pai ed-end me agenome eads om indi idual da ase s we e mapped e sus all assembled me a- genome con igs wi h Bow ie 2 ( 2.2.4) [51]inend- o-end mode applying he op ion ‘sensi i e’. To con e SAM o BAM, so he alignmen ile and calcula e ead mapping s a is ics SAM ools ( 1.0) [52] was used. Fu he mo e, o p edic genes on assembled con igs la ge han 1 kb, he gene p edic ion ool P odigal .2.6.0 [53] was applied. P e- dic ed p o ein sequences we e compa ed o NCBI’sda a- base using he BLASTP mode o DIAMOND [54]. The esul ing ou pu ile was loaded in o MEGAN5 [55] o axonomic classi ica ion o each gene sequence. In he ol- lowing binning s ep, he abundance p o ile and he e anu- cleo ide equencies we e used o bin con igs in o me agenome-assembled genomes (MAGs) wi h Me aBAT ( 0.21.3) [56]. Mapping o he eads was always pe o med on all con igs, including he con igs o he MAGs and he con igs which we e no binned. Subsequen ly, comple e- ness, con amina ion, and s ain he e ogenei y o he MAGs we e es ima ed wi h CheckM ( 1.0.4) [22], using se s o clade-speci ic single-copy ma ke genes. Mic obial me a ansc ip ome lib a y p epa a ion and NGS The o al mic obial RNA om wo sub-samples ( om he same silage bio ilm as o he DNA ex ac ion) was ex ac ed applying he RNeasy Mini Ki (Qiagen, Hilden, Ge many) acco ding o he manu ac u e ’s guidelines. In o al, wo echnical eplica es we e p ocessed o each o wo pa allel hyd oly ic eac o communi ies yielding eigh samples o he mesophilic sys em and eigh samples o he he mophilic sys em (Addi ional ile 7). Subsequen ly, he RNA was pu i ied using he Ambion® Tu bo DNA- ee Ki (The moFishe , Ge many). Ribosomal RNA was deple ed using he Ribo-Ze o™ RNA Remo al Ki o Bac e ia (Illumina, Madison, USA) acco ding o he manu ac u e ’sin- s uc ions. The emaining mRNA ansc ip s we e agmen ed o app oxima ely 550 bp agmen s using a ocused-ul asonica o (Co a is M220, Wobu n, USA). cDNA lib a ies o Illumina sequencing we e cons uc edusing heT uSeqRNALib a yP epKi 2 (Illumina, Eindho en, Ne he lands). The esul ing cDNA lib a ies we e sequenced on he Illumina HiSeq 1500 machine using he Illumina HiSeq Rapid SBS Ki 2 (Illumina, Eindho en, Ne he lands) o gene a e 2 × 100 bp pai ed-end eads. Me agenome and me a ansc ip ome sequence analysis Pai ed end me agenome and me a ansc ip ome eads we e mapped e sus all assembled me agenome con igs wi h Bow ie2 [51] in end- o-end mode wi h op ion sen- si i e. A e ead mapping SAM ools [52] was used o il- e he esul ing BAM iles o uniquely mapped eads. Reads we e classi ied as uniquely mapped eads wi h a unique genomic loca ion i and only i hey could no be aligned o ano he loca ion wi h a highe o same map- ping quali y. Me agenome as well as me a ansc ip ome eads ha could be aligned o assembled MAG’s we e quan i ied wi h he HTSeq-coun p og am [57] o ge an es ima e o MAG abundance and o e all MAG exp es- sion, espec i ely. To accoun o di e en MAG abun- dance, aw me a ansc ip ome ead coun s o each MAG plus one (pseudocoun ) we e di ided by he MAG’s aw me agenome ead coun plus one and ounded o in ege alues. The esul ing coun s o each MAG se ed as inpu o DESeq2 [58] o pai wise de- ec ion and quan i ica ion o di e en ially abundan and ansc ip ionally ac i e MAGs, espec i ely. Fo DESeq2 pa ame iza ion, a be a p io and disabled Cook dis ance cu o il e ing was used. All o he pa ame e s emained unchanged. Fold change es ima es, p- alues, and egula - ized log- ans o med ( log) coun s OF (1) me agenome ead coun s and (2) me a ansc ip ome ead coun s no - malized o di e en MAG abundances as desc ibed abo e we e emplyed o assess p ope sample esp. epli- ca e clus e ing wi h (PCA, mul idimensional scaling (MDS)), and hie a chical clus e ing. In addi ion, olcano plo s we e used o iden i y signi ican ly di e en ially exp essed MAGs. Simila compa a i e analyses we e pe o med assuming equal MAG abundances. Phylogene ic and unc ional analysis o he me agenome- assembled genomes The Genome Taxonomy Da abase oolki [59] was used o assign objec i e axonomic classi ica ions o bac e ial and a chaeal genomes. Each MAG was de ined as single ope a ional axonomic uni (OTU). Fo MAGs wi h comple eness alues o mo e han 50% and a con amin- a ion a e less han 10% (Addi ional ile 4), analysis o he gene ic po en ial was pe o med using he EMGB [60] anno a ion sys em including KEGG pa hway map- ping and DIAMOND ool [54]. MAGs ha mee he c i- e ia men ioned abo e we e subsequen ly analyzed ega ding hei ansc ip ional ac i i ies using he EMGB anno a ion sys em again. To p edic genes encoding ca bohyd a e-ac i e en- zymes, he ca bohyd a e-ac i e enzyme da abase (CAZy) Maus e al. En i onmen al Mic obiome (2020) 15:7 Page 18 o 21 anno a ion web se e dbCAN 7 [24] was used. The genes encoding enzymes ac ing di ec on cellulose we e iden i ied by he p esence o coding egions o ype one o wo docke in o cohesin modules, among o he s. Supplemen a y in o ma ion Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10. 1186/s40793-020-00354-x. Addi ional ile 1. S a is ics o 16S RNA gene sequence analysis. Addi ional ile 2. Rela i e abundances o mos abundan 16S RNA gene sequences. Addi ional ile 3. S a is ics o he ob ained and p ocessed me agenome and me a ansc ip ome sequences. Addi ional ile 4. Taxonomic a ilia ions o me agenome-assembled ge- nomes (MAGs) o his s udy o igina ing om HR bio ilms. Addi ional ile 5. Analysis o he key enzymes o AD pa hways in me agenome-assembled genomes (MAGs). The gene ic de e minan s we e ca ego ized acco ding o he ou s ages o he AD p ocess, namely hyd olysis, acidogenesis, ace ogenesis and me hanogenesis as desc ibed p e iously [23]. Addi ional ile 6. Hie a chical clus e ing o abundance alues o 78 selec ed me agenome-assembled genomes (MAGs) de ec ed in HR bio- ilms a mesophilic and he mophilic p ocess empe a u e a o ganic loading a e (OLR) o 500 g esp. 1500 g yeg ass silage as deduced om ansc ip ome da a. Addi ional ile 7. Expe imen al se up and sampling scheme. Abb e ia ions AD: Anae obic diges ion; AF: Anae obic il e ; ANI: A e age nucleo ide sequence iden i y; CAZymes: Ca bohyd a e-ac i e enzymes; CO 2 : Ca bon dioxide; COD: Chemical oxygen demand; d: day; FM: F esh mass; GH: Glycosyl hyd olase; HR: Hyd olysis eac o ; HT: High- h oughpu ; kg: kilog am; kg VS : kilog ams ola ile subs ances; L: Li e ; L N : Li e no malized; M: Mesophilic; MAG: Me agenome assembled genomes; MG: Mic obial me agenome da ase ; MT: Mic obial me a ansc ip ome da ase ; OLR: O ganic loading a es; OTU: Ope a ional axonomic uni ; SAOB: Syn ophic ace a e- oxidizing bac e ia; T: The mophilic; TPM: T ansc ip s pe million; TRFLP: Te minal es ic ion agmen leng h polymo ophism; VFA: Vola ile a y acids; VS: Vola ile subs ances Acknowledgemen s The au ho s g a e ully acknowledge he highly aluable echnical suppo o M. Felgen eu, M. Jäkel, K. Mund , and G. Rehde. The bioin o ma ics suppo o he BMBF- unded p ojec ‘Biele eld-Gießen Cen e o Mic obial Bioin o - ma ics - BiGi (g an no. 031A533)’wi hin he Ge man Ne wo k o Bioin o - ma ics In as uc u e (de.NBI) is also g a e ully acknowledged. ASz and A.S. acknowledge unding om he Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og am unde g an ag eemen No. 818431 (SIMBA). This ou pu e lec s only he au ho ’s iew and he Resea ch Execu i e Agency (REA) can- no be held esponsible o any use ha may be made o he in o ma ion con ained he ein. Au ho s’con ibu ions IM p epa ed he 16S RNA gene amplicon lib a ies o sequencing, analyzed he axonomic p o iles o bio ilm communi ies, analyzed and in e p e ed me agenome and me a ansc ip ome da a, pe o med he compa a i e MAG analyses, coo dina ed d a ing and d a ed co esponding pa s o he manusc ip . MK pa icipa ed in da a analysis and isualiza ion, and d a ed pa s o he in oduc ion, esul s, discussion, and conclusions sec ions. JD conduc ed all AD expe imen s, he sampling o diges a es and p ocess liquids, DNA and RNA ex ac ion om bio ilm samples, and TRFLP analysis. JD con ibu ed also o AD da a analysis and e ised he manusc ip . YS also pe o med DNA and RNA ex ac ion om bio ilm samples, p epa ed he me agenome and me a ansc ip ome sequencing lib a ies, and e ised he manusc ip . DW and JB de e mined he phylogene ic ela ionship be ween he MAGs, con ibu ed o he esul s and discussion sec ion, and e ised he manusc ip . MB pe o med he compa a i e me agenome and me a ansc ip ome analysis, con ibu ed o he ma e ial and me hods sec ion, and e ised he manusc ip . CJ pa icipa ed in AD expe imen s and co esponding da a analysis, and e ised he manusc ip . KW pa icipa ed in me a ansc ip ome da a analysis, and e ised he manusc ip . MR ca ied ou he axonomic classi ica ion o he mic obial communi ies, con ibu ed o he esul s sec ion, and e ised he manusc ip . AR pa icipa ed in AD expe imen s and TRFLP analysis and co esponding da a analysis, and e ised he manusc ip . AP pa icipa ed in he design o his s udy, con ibu ed o he esul s and discussion sec ions, and e ised he manusc ip . ASz and CH ca ied ou he axonomic classi ica ion o he mic obial communi ies based on me agenome and me a ansc ip ome da a, pe o med he me agenome assembly and binning, pa icipa ed in he compa a i e me agenome and me a ansc ip ome analysis, con ibu ed o he esul s and discussion sec ion, and e ised he manusc ip . ASz, AS, and MK concei ed he s udy, pa icipa ed in manusc ip coo dina ion, o ganized hi d-pa y unding, su- pe ised all biological and bioin o ma ic da a analyses, con ibu ed o d a - ing o he manusc ip and e ised he manusc ip . All au ho s ead and app o ed he inal manusc ip . Funding This wo k was pa o he join p ojec BIOGAS-MARKER suppo ed by he Ge man Fede al Minis y o Educa ion and Resea ch (BMBF), g an nos. 03SF0440A and 03SF0440C. A ailabili y o da a and ma e ials Sequence da ase s we e deposi ed in he Eu opean Nucleo ide A chi e (ENA) unde he Biop ojec accession numbe s PRJEB27769 (me agenome da ase s), E-MTAB-7533 (me a ansc ip ome da ase s), PRJEB30260 (16S RNA gene amplicon sequences). E hics app o al and consen o pa icipa e No applicable. Consen o publica ion No applicable. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. 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