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)
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* 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
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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.
Au ho de ails
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.
2
Depa men Bioenginee ing, Leibniz Ins i u e o Ag icul u al Enginee ing
and Bioeconomy (ATB), Max-Ey h-Allee 100, 14469 Po sdam, Ge many.
3
Helmhol z Cen e o In ec ion Resea ch, Mic obial In ec ion Biology /
Expe imen al Immunology, Inho ens asse 7, 38124 B aunschweig, Ge many.
4
Depa men Bioin o ma ics and Sys ems Biology, Jus us-Liebig Uni e si y
Gießen, Hein ich-Bu -Ring 58, 35392 Giessen, Ge many.
5
Facul y o
Technology, Biele eld Uni e si y, Uni e si ä ss . 25, 33615 Biele eld, Ge many.
Recei ed: 20 Sep embe 2019 Accep ed: 14 Feb ua y 2020
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