Impact of Oxygen and Pesticides on Microbial Cellulose Degradation in Aerated Agricultural Soils: A Microscaled Analysis of Processes and Prokaryotic Populations
Full text
Impac o Oxygen and Pes icides on Mic obial
Cellulose Deg ada ion in Ae a ed Ag icul u al
Soils: A Mic oscaled Analysis o P ocesses
and P oka yo ic Popula ions
Disse a ion
To ob ain he Academic Deg ee
Doc o e um na u alium
(D . e . na .)
Submi ed o he Facul y o Biology, Chemis y and Ea h Sciences
o he Uni e si y o Bay eu h
by
S e anie Schellenbe ge
Bay eu h, No embe 2011
The p esen s udy was p epa ed be ween No embe 2007 and No embe 2011 a
he Depa men o Ecological Mic obiology a he Uni e si y o Bay eu h unde he
di ec ion o P o . Ha old L. D ake.
The wo k was inancially suppo ed by he Deu sche Fo schungsgemeinscha (DFG
Ko2912/3-1) and he Uni e si y o Bay eu h.
Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und
Geowissenscha en de Uni e si ä Bay eu h genehmig en Disse a ion zu
E langung des akademischen G ades eines Dok o s de Na u wissenscha en (D .
e . na ).
Disse a ion einge eich : 11.11.2011
Zulassung du ch die P ü ungskommission: 28.02.2012
Wissenscha liches Kolloquium: 24.04.2012
Am ie ende Dekan: P o . D . Bea e Lohne
P ü ungsausschuss:
P o . Ha old L. D ake (E s gu ach e )
P o . D . O win Meye (Zwei gu ach e )
P o . D . Angelika Mus oph (Vo si z)
P o . D . Heike Feldhaa
P o . D . Egbe Ma zne
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TABLES .................................................................................................. I
FIGURES .............................................................................................. III
EQUATIONS .......................................................................................... V
ABBREVIATIONS ................................................................................. VI
1. INTRODUCTION ............................................................................. 1
1.1. Ca bon Flow h ough Te es ial Ecosys ems ........................................... 1
1.2. Cellulose – a Majo Polysaccha ide in Soils .............................................. 2
1.2.1. Chemical S uc u e o Cellulose and i s Enzyma ic Hyd olysis ....... 3
1.2.1.1. Non-Complexed Enzyme Sys ems ................................................ 5
1.2.1.2. Complexed Enzyme Sys ems (Cellulosomes) ............................... 6
1.2.1.3. Ex acellula Cellulose Dis up ing Complex in G am-nega i e
Bac e ia .......................................................................................... 6
1.2.2. Biological Deg ada ion o Cellulose in Soils ..................................... 6
1.3. Di e si y o Cellulose-Deg ading O ganisms ............................................. 9
1.3.1. Euka yo es ............................................................................................ 9
1.3.2. P oka oy es ........................................................................................ 10
1.4. De ec ion o Cellulose-Deg ading O ganisms ......................................... 12
1.5. Cellulose-Deg ading Mic oo ganisms in Soil Ha e Simila
Ecological Niches ....................................................................................... 12
1.6. En i onmen al and An h opogenic Fac o s In luence Bac e ial
Cellulose Deg ada ion in Ag icul u al Soil ............................................... 14
1.6.1. Changing A ailabili y o O
2
Induces Redox Changes ..................... 14
1.6.2. Pes icides In luence Biological P ocesses ...................................... 15
1.7. Hypo heses and Objec i es ....................................................................... 16
2. MATERIAL AND METHODS ........................................................ 17
2.1. Chemicals, Gases, and Media.................................................................... 17
2.2. Sampling Si e and Soil Cha ac e is ics .................................................... 18
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2.3. Soil Mic ocosms ......................................................................................... 19
2.3.1. T ea men s o S able Iso ope P obing (SIP) ................................... 20
2.3.1.1. Supplemen a ion o Cellulose ...................................................... 20
2.3.1.2. Supplemen a ion o Cellobiose and Glucose ............................... 21
2.3.2. T ea men s in Sel -Cons uc ed Incuba ion Chambe s .................. 21
2.3.2.1. Design o Incuba ion Chambe s .................................................. 21
2.3.2.2. Incuba ion Condi ions .................................................................. 22
2.3.3. T ea men s o Resol e Impac o Pes icides on he
Deg ada ion o Saccha ides ............................................................. 23
2.3.3.1. Pes icides .................................................................................... 23
2.3.3.2. Impac o Ben azon, MCPA, and Nonylphenol on he
Deg ada ion o Cellobiose ........................................................... 26
2.3.3.3. Impac o Ben azon, MCPA, and Nonylphenol on he
Deg ada ion o Cellulosic Pape Shee s ...................................... 26
2.3.3.4. Quan i ica ion o Pes icide E ec s on P ocesses Linked o he
Deg ada ion o Cellulose and Cellobiose .................................... 27
2.4. Analy ical Me hods .................................................................................... 27
2.4.1. D y Weigh and G a ime ic Wa e Con en o Soil ........................ 27
2.4.2. To al Ca bon and Ni ogen Con en ................................................. 27
2.4.3. Soil Tex u e and Soil Type ................................................................ 28
2.4.4. pH ........................................................................................................ 28
2.4.5. Redox Po en ial .................................................................................. 28
2.4.6. Gases .................................................................................................. 28
2.4.7. Soluble O ganic Compounds ........................................................... 31
2.4.8. Cellulose ............................................................................................. 32
2.4.9. Pes icides ........................................................................................... 32
2.4.10. Ino ganic Compounds ....................................................................... 33
2.4.10.1. Ni a e (NO
3-
) and To al Amoun s o Ammonium (NH
4+
), I on,
Manganese, and Sulpha e .......................................................... 33
2.4.10.2. Fe ous I on (Fe
2+
) ...................................................................... 33
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2.5. Molecula Me hods ..................................................................................... 33
2.5.1. Ex ac ion o Nucleic Acids ............................................................... 33
2.5.2. Sepa a ion o DNA and RNA ............................................................. 34
2.5.2.1. Solid Phase Columns................................................................... 34
2.5.2.2. Enzyma ic Sepa a ion .................................................................. 34
2.5.3. Pu i ica ion and P ecipi a ion o Nucleic Acids ............................... 34
2.5.3.1. Isop opanol/Sodium Chlo ide P ecipi a ion .................................. 34
2.5.3.2. Gel Ex ac ion .............................................................................. 34
2.5.3.3. Fil e Pla es .................................................................................. 35
2.5.4. Quali y Con ol and Quan i ica ion o Nucleic Acids ...................... 35
2.5.4.1. Spec opho ome y ...................................................................... 35
2.5.4.2. Pico-/RiboG een-Based Quan i ica ion ........................................ 35
2.5.5. Aga ose Gel Elec opho esis ............................................................ 36
2.5.6. RNA S able Iso ope P obing (RNA SIP) .......................................... 36
2.5.6.1. Densi y G adien Cen i uga ion ................................................... 37
2.5.6.2. F ac iona ion o G adien s ............................................................ 38
2.5.6.3. Measu emen o he Densi y o he F ac ions .............................. 38
2.5.6.4. RNA P ecipi a ion ........................................................................ 39
2.5.7. Re e se T ansc ip ion o RNA in o cDNA ........................................ 39
2.5.8. In Vi o T ansc ip ion o DNA in o RNA ............................................ 39
2.5.9. Polyme ase Chain Reac ion (PCR) ................................................... 39
2.5.9.1. P ime s and The mal P o ocols ................................................... 40
2.5.9.2. 16S/18S RNA Genes .................................................................. 42
2.5.9.3. Clone Inse Sequences ............................................................... 43
2.5.10. Quan i a i e PCR (qPCR) ................................................................... 44
2.5.10.1. QPCR P ime s and The mal P o ocols ........................................ 45
2.5.10.2. P epa a ion o Quan i a i e DNA S anda ds ................................ 48
2.5.10.3. E alua ion o he Speci ici y o Assays in Soil Samples ............... 49
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2.5.10.4. Co ec ion o PCR Inhibi ion and Calcula ion o T ansc ip
Numbe s ...................................................................................... 49
2.5.11. Te minal Res ic ion F agmen Leng h Polymo phism ( RFLP)
Analysis .............................................................................................. 51
2.5.11.1. Mung Bean Endonuclease Diges ion .......................................... 52
2.5.11.2. Res ic ion Diges ion ................................................................... 52
2.5.11.3. Dena u ing Polyac ylamide Gelelec opho esis (PAGE) ............. 52
2.5.11.4. Analysis o RFLP P o iles ........................................................... 53
2.5.12. Cons uc ion o Gene Lib a ies ........................................................ 53
2.5.12.1. Liga ion ........................................................................................ 53
2.5.12.2. T ans o ma ion ............................................................................ 54
2.5.12.3. Blue/whi e Sc eening o Clones .................................................. 55
2.5.13. Sequencing ........................................................................................ 55
2.5.14. Gene Sequence Analysis .................................................................. 56
2.5.14.1. P ocessing o Sequences De i ed om S able Iso ope
P obing Expe imen s ................................................................... 56
2.5.14.2. Ra e ac ion Analysis .................................................................... 57
2.5.14.3. Calcula ion o Phylogene ic T ees ............................................... 57
2.5.14.4. Deposi ion o Sequences in O icial Da abases ........................... 57
2.6. S a is ics ..................................................................................................... 58
2.6.1. Mean Value, S anda d De ia ion, and E o P opaga ion .............. 58
2.6.2. Tes o Signi icance ( -Tes ) .............................................................. 59
3. RESULTS ...................................................................................... 60
3.1. Deg ada ion o [
12
C]- and [
13
C]-Cellulose, -Cellobiose, and -Glucose
unde Oxic and Anoxic Condi ions .......................................................... 60
3.2. Iden i ica ion o Ac i e P oka yo es in [
13
C]-Cellulose, -Cellobiose,
and -Glucose Supplemen ed T ea men s by RNA S able Iso ope
P obing ....................................................................................................... 63
3.2.1. Dis ibu ion o RNA in G adien F ac ions and Selec ion o
’Ligh ’ and ’Hea y’ F ac ions o Molecula Analyses .................... 63
3.2.2. TRFLP P o iles o A chaeal 16S RNA cDNA Sequences .............. 65
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3.2.3. TRFLP P o iles o Bac e ial 16S RNA cDNA Sequences ............... 67
3.2.3.1. Compa ison o ‘Hea y’ and ‘Ligh ’ F ac ions in Oxic and
Anoxic [
12
C]- o [
13
C]-T ea men s ................................................. 67
3.2.3.2. Iden i ica ion o Labeled RFs by Compa ison o ‘Hea y’
F ac ions be ween [
12
C]- and [
13
C]-T ea men s, and
Phylogene ic A ilia ion ................................................................. 71
3.2.3.2.1. Bac e ia ha Inco po a ed [
13
C]-Ca bon in Oxic T ea men s .... 71
3.2.3.2.2. Bac e ia ha Inco po a ed [
13
C]-Ca bon in Anoxic
T ea men s ................................................................................ 76
3.2.4. Bac e ial 16S RNA cDNA Gene Lib a ies o ‘Hea y’ F ac ions ..... 81
3.2.4.1. Ra e ac ion and Co e age ........................................................... 81
3.2.4.2. G ouping o Sequences in o Family-le el OTUs and hei
Phylogene ic A ilia ion ................................................................. 84
3.2.4.2.1. Labeled Taxa ............................................................................ 84
3.2.4.2.2. Non-labeled Taxa ..................................................................... 88
3.3. Labeled Euka yo es .................................................................................... 90
3.4. Design o Family-le el qPCR Assays ........................................................ 92
3.5. E ec o Pes icides on he Deg ada ion o Cellulose and Cellobiose ... 96
3.5.1. In Si u- ele an (‘low’) Concen a ions o Pes icides ...................... 97
3.5.2. Ele a ed (‘high’) Concen a ions o Pes icides ............................... 98
3.5.3. 16S RNA Con en o Bac e ial Taxa ............................................... 103
3.6. E ec o Fluc ua ing A ailabili ies o O
2
on he Oxic and Anoxic
Deg ada ion o CMC and Cellobiose, and E alua ion o Me abolic
Responses o Saccha ide-u ilizing P oka yo es .................................... 106
3.6.1. E ec o he Deg ada ion o CMC and Cellobiose on Redox
Po en ials, and he Fo ma ion o Deg ada ion P oduc s .............. 106
3.6.2. 16S RNA Con en o P oka yo ic Taxa .......................................... 109
4. DISCUSSION ...............................................................................114
4.1. Ae obic and Anae obic Deg ada ion o Saccha ides in An Ae a ed
Ag icul u al Soil ........................................................................................ 114
4.2. Ac i e Saccha ide-Deg ading P oka yo ic Communi ies ...................... 118
4.2.1. Key Taxa ha Deg aded Saccha ides unde Oxic Condi ions ..... 119
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4.2.2. Key Taxa ha Deg aded Saccha ides unde Anoxic Condi ions 121
4.2.3. Labeled Taxa ha a e o Mino Impo ance o he Deg ada ion
o Saccha ides ................................................................................. 123
4.2.4. A La ge Uncul u ed Sub-Communi y is Selec i ely Ac i a ed
du ing he Ae obic and Anae obic Deg ada ion o Cellulose ...... 125
4.2.5. Deg ada ion o Cellulose is a S able Communi y Func ion in
he In es iga ed Soil ........................................................................ 126
4.3. Di e si y o Me abolic Ac i e bu no [
13
C]-Labeled Bac e ia ............... 127
4.4. Di e si y o Ac i e Euka yo es ................................................................ 128
4.5. Pes icides ha e Mino E ec s on he Me abolism o Cellulose-
Deg ading Communi ies .......................................................................... 130
4.6. Conclusions and Model ........................................................................... 132
4.7. Limi a ion o he Applied Me hods ......................................................... 134
4.8. Fu u e Pe spec i es ................................................................................. 136
5. SUMMARY .................................................................................. 137
6. ZUSAMMENFASSUNG .............................................................. 139
7. REFERENCES ............................................................................ 141
8. ACKNOWLEDGEMENTS ........................................................... 181
9. PUBLICATIONS AND PRESENTATIONS .................................. 182
9.1. Publica ions and Manusc ip s ................................................................ 182
9.2. P esen ions wi h Abs ac s ..................................................................... 182
9.3. P esen a ions wi hou Abs ac s ............................................................ 183
10. DECLARATION .......................................................................... 184
APPENDICES ...................................................................................... A.I
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I
TABLES
Table 1. Lis o bac e ial phyla including cul u ed ae obic and anae obic celluloly ic
species. ............................................................................................................. 11
Table 2. LB medium. ....................................................................................................... 17
Table 3. SOC medium ..................................................................................................... 18
Table 4. Soil cha ac e is ics ............................................................................................. 19
Table 5. Pes icides ha we e es ed. ............................................................................... 24
Table 6. Concen a ion o pes icides in ea men s. ......................................................... 25
Table 7. Pa ame e s o GC measu emen s. .................................................................... 29
Table 8. Bunsen solubili y coe icien s o ca bon dioxide, me hane, and molecula
hyd ogen a di e en empe a u es. ................................................................... 30
Table 9. Pa ame e s o HPLC sys em o soluble o ganic compounds. ........................... 31
Table 10. Pa ame e s o pes icide measu emen s by HPLC. ............................................. 32
Table 11. P ime s o ampli ica ion o 16S RNA genes o Bac e ia and A chaea, and o
sequences inse ed in o he pGEM-T ec o o clones. ...................................... 40
Table 12. Chemical composi ion o PCR eac ions ............................................................ 41
Table 13. The mal p o ocols o ampli ica ion o 16S RNA genes and clone inse
sequences. ........................................................................................................ 42
Table 14. P ime s o ampli ica ion o 18S RNA genes o Euka ya. .................................. 43
Table 15. P ime s o quan i ica ion o 16S RNA genes o Bac e ia, A chaea and
amily-le el axa, and o inhibi ion co ec ion by qPCR. .................................... 45
Table 16. Chemical composi ion o qPCR eac ions .......................................................... 47
Table 17. The mal p o ocols o ampli ica ion o 16S RNA genes and clone inse
sequences. ........................................................................................................ 48
Table 18. Liga ion eac ion mix. ......................................................................................... 54
Table 19. Iden i ica ion and occu ence o labeled RFs in oxic [
13
C]- ea men s. ............... 75
Table 20. Iden i ica ion and occu ence o labeled RFs in anoxic [
13
C]- ea men s. ........... 80
Table 21. Cha ac e is ics o 16S RNA cDNA gene lib a ies o ’hea y’ ac ions o oxic
and anoxic [
13
C]-cellulose, [
13
C]-cellobiose, and [
13
C]-glucose ea men s. ......... 82
Table 22. Rela i e abundances o labeled OTUs ob ained om 16S RNA cDNA gene
lib a ies om ‘hea y’ ac ions o [
13
C]- ea men s and hei phylogene ic
a ilia ion. ........................................................................................................... 85
Table 23. Non-labeled amily-le el axa ob ained om 16S RNA cDNA gene lib a ies
om ‘hea y’ ac ions o [
13
C]- ea men s, hei ela i e abundance o e all
16S RNA cDNA sequences, and celluloly ic isola es. ....................................... 89
Table 24. Iden i ica ion and occu ence o labeled euka yo ic RFs in [
13
C]-cellulose
ea men s ......................................................................................................... 91
Table 25. Expe imen ally es ablished pa ame e s o speci ic qPCR measu emen s o
amily-le el assays............................................................................................. 93
Table 26. Speci ici y o qPCR assays ................................................................................ 95
A
BBREVIATIONS
VIII
N 1. ni ogen;
2. o al numbe o analysed sequences
n 1. amoun o subs ance,
2. numbe o eplica es/conside ed alues
n.a. non-applicable
n.i. 1. no iden i ied
2. no inhibi ion
N
A
A ogad o cons an
NaCl sodium chlo ide
NaOH sodium hyd oxide
N
bases
leng h o amplicon
NCBI Na ional Cen e o Bio echnology In o ma ion
n
gas
amoun o gas in he gaseous phase
n
gel,p
amoun o physically dissol ed gas
n
ges
o al amoun o gas
n
ges,c
amoun o chemically dissol ed gas
nm nanome e
nM nanomola ; nanomole pe li e
no. numbe
OD
660
op ical densi y a 660 nm
OTU ope a ional axonomic uni
p
1
s anda dized ai p essu e
PAGE Polyac ylamide gel
p
ak
ac ual ai p essu e
PCR polyme ase chain eac ion
PCR-H
2
O pa icle- ee au ocla ed wa e
PEG polye hylene glycol
Pg pe ag amm
pg picog amm
pH he nega i e decimal loga i hm o he hyd ogen ion ac i i y in a
solu ion
pheA gene encoding a phenol hyd olyase
pk
a
acid dissocia ion cons an
PLFA phospholipid a y acids
pmoA gene encoding he pa icula e me hane monooxygenase
ppm pa s pe million
p
ü
o e p essu e in incuba ion lasks
qPCR quan i a i e polyme ase chain eac ion
R e e se p ime
R
2
s abili y index
RDP Ribosomal Da abase P ojec
RID e ac i e index de ec o
RNA ibonucleic acid
RNAse ibonuclease
pm ounds pe minu e
RNA ibosomal ibonucleic acid
RT-PCR e e se ansc ip ion PCR
S s anda d de ia ion
Seq. sequence
S
i
numbe o expec ed OTUs
SIP s able iso ope p obing
A
BBREVIATIONS
IX
SOC supe op imal b o h; medium
sp. species
SQ s a ing quan i y
1. on (=1,000 kg);
2. alue ha implies s a is ical signi icance
T
1
s anda dized empe a u e
TAE is-acea e-EDTA; bu e
T
ak
ac ual empe a u e
Taq he mos able DNA polyme ase isola ed om The mus aqua icus
TaqI Res ic ion endonuclease isola ed om The mus aqua icus
TBE is-bo a -EDTA; bu e
TCD he mal conduc i i y de ec o
TEMED N,N,N,N- e ame hyle hylendiame
emp. empe a u e
T
m
basic mel ing empe a u e
RF e minal es ic ion agmen
RFLP e minal es ic ion agmen leng h polymo phism
T is is(hyd oxyme hyl)-aminome hane
U uni
UFZ Helmhol z-Cen e o En i onmen al Resea ch
UV ul a iole
ν a iance
V 1. olume;
2. ol
/ olume pe olume
V
1
s anda dized mola gas olume
V
ak ,mol
mola olume o gas unde ac ual condi ions
V
l
olume o liquid phase
V
gas
olume o he gas phase
VWD a iable wa eleng h de ec o
w/ weigh pe olume
W
g
g a ime ic wa e con en
X-Gal 5-B omo-4-chlo o-3-indolyl-β-D-galac opy anoside
I
NTRODUCTION
1
1.
I
NTRODUCTION
1.1. Ca bon Flow h ough Te es ial Ecosys ems
Con e sion o biomass o ca bon dioxide (CO
2
) and me hane (CH
4
) in e es ial
ecosys ems is a subs an ial pa o global ca bon cycling (Amundson 2001; Be ns ein
e al. 2007). The global ca bon cycle is a long- e m balance be ween a mosphe ic
ca bon, and e es ial and aqua ic ca bon sinks and ca bon sou ces (Dumonceaux
2005; Falkowski e al. 2000). A mosphe ic ca bon occu s p ima ily as ca bon dioxide
and i s concen a ion inc eased du ing he las wo cen u ies o 385 ppm (Lal 2008).
The o al amoun o ca bon in he a mosphe e is abou 780 Pg (Figu e 1) and
inc eases abou 4.1 Pg yea
-1
due o an h opogenic ac i i ies (Amundson 2001;
Be ns ein e al. 2007; Lal 2008).
Figu e 1. Schema ic scheme o he global ca bon low.
Global ca bon pools and
exchange o ca bon wi h he a mosphe e a e displayed. Numbe s ep esen es ima ed
amoun s o ca bon in Pg yea
-1
. Nega i e numbe s indica e ca bon consumed by he
ecosys em ype. Posi i e numbe s indica e ca bon ha is eleased o he a mosphe e.
Values a e acco ding o Lal 2008.
An h opogenic ac i i ies like ossil uel bu ning (7.5 Pg ca bon yea
-1
) and
in ensi e land use (~1.6 Pg ca bon yea
-1
) emi mo e han 9 Pg ca bon yea
-1
(Figu e 1; Lal 2008). Oceans ha ake up 2.5 Pg o an h opogenic ca bon dioxide
and e es ial ecosys ems a e impo an sinks o a mosphe ic ca bon dioxide
(Schimel 1995). I is es ima ed ha ~120 Pg o ca bon yea
-1
a e ixed by plan s in
he o m o a mosphe ic ca bon dioxide and s o ed in plan biomass (Dumonceaux
2005). Howe e , 2.6 Pg mo e ca bon is ixed by unknown e es ial sinks (Lal 2008).
Au o ophic espi a ion by plan s (~60 Pg yea
-1
) and dis u bances, e.g., wild i es,
I
NTRODUCTION
2
con ibu e o he elease o la ge amoun s o ca bon dioxide back in o he
a mosphe e (Falkowski e al. 2000; Lal 2008). Ano he 60 Pg ca bon yea
-1
is
e u ned o he a mosphe e by biological decomposi ion o plan biomass. Soil
o ganisms (e.g., bac e ia, ungi, p o is s) ae obically and anae obically me abolize
plan -de i ed ca bon, p oduce ca bon dioxide, and close he global ca bon cycle
(Dumonceaux 2005; Lal 2008). An impo an cha ac e is ic o soil is ha i has he
capaci y o s o e la ge amoun s o a mosphe ic ca bon dioxide. A mosphe ic ca bon
dioxide is pho osyn he ically ixed and pu in o he soil as ecalci an plan -de i ed
compounds (Amundson 2001; Lal 2008). Soils ake up 1.7 Pg o an h opogenic
ca bon yea
-1
(Figu e 1), making hem o a key ecosys em conce ning he global
ca bon budge . The amoun o ca bon ha is s o ed in soil is de e mined by a lo o
di e se ac o s, e.g., clima e, opog aphic posi ion, empe a u e, po en ial soil bio a,
o human ac i i ies (Amundson 2001). In con as , a signi ican pa o ca bon dioxide
ha is e u ned back in o he a mosphe e p ima ily depends on he ac i i y o soil
o ganisms ha decompose plan ma e and plan -de i ed ca bon sou ces (Falkowski
e al. 2000). Con e sion o na u al o es s, g asslands, and we lands o ag icul u al
ecosys ems (i) dec eases he amoun o o ganic ca bon ha is s o ed in soil and (ii)
inc eases he amoun o ca bon dioxide ha is eleased in o he a mosphe e
(Schimel 1995; Schlesinge and And ews 2000). Especially illage by c op a ming
acili a es ca bon loss om soil by imp o ing condi ions o mic obial decomposi ion
o o ganic ca bon. C op a ming leads o be e soil ae a ion and highe soil
empe a u es, and simul aneously educes he inpu o plan ma e ial in o soil
compa ed o he na i e ege a ion (Amundson 2001; Schlesinge and And ews
2000). Fe iliza ion o use o pes icides in ag icul u e in luences ca bon low in
ag icul u al soils (Lal 2008; Schlesinge and And ews 2000) and migh also impac on
soil o ganisms ha a e in ol ed in he decomposi ion o plan -de i ed o ganic ma e .
Hence, soil o ganisms ha decompose plan s a e key playe s in he global ca bon
cycle since hey d i e a subs an ial pa o ca bon low h ough e es ial
ecosys ems.
1.2. Cellulose – a Majo Polysaccha ide in Soils
Biological decomposi ion o plan ma e ial o ca bon dioxide is a complex
p ocess ha s a s a s uc u al polyme s. The lignocellulose complex o ms he cell
wall o plan s and is composed o lignin, cellulose, hemicelluloses, and o he
polyme s (Chang 2007; Kuma e al. 2008a; Malhe be and Cloe e 2002).
Lignocellulose is he mos abundan and enewable ene gy sou ce on ea h.
Te es ial plan s p oduce as much biomass as equi alen o wo- hi ds o he wo ld’s
ene gy equi emen pe yea (Demain e al. 2005). The polysaccha ide cellulose is
he majo componen o lignocellulose and cons i u es 35 – 50% o he d y weigh o
plan s o li e om deciduous and coni e ous ees (Kögel-Knabne 2002; Lynd e al.
2002). Dependen on he s age o g ow h, co on can be composed o up o 95% o
cellulose (Abidi e al. 2010). Howe e , mino amoun s o cellulose a e ound in cell
wall polyme s o ce ain bac e ia, ungi, slime molds, and amoebae (Coughlan 1985;
Lynd e al. 2002; Tomme e al. 1995). I is es ima ed ha cellulose is p oduced on
ea h by pho osyn hesis a leas a a a e o 10
9
– 10
10
yea
-1
and ha a simila
amoun is deg aded by celluloly ic, i.e., cellulose-deg ading, and saccha oly ic
o ganisms, i.e., o ganisms ha me abolize p oduc s o cellulose hyd olysis like
cellobiose o glucose (Coughlan 1985; Hen issa 1994). S uc u e and hyd olysis o
cellulose has been ex ensi ely s udied due o he inc easing in e es s o
bio echnologis s and o he s o indus ial use o his biopolyme (e.g., Baye e al.
I
NTRODUCTION
3
2008; Beguin and Aube 1994; Be ghem e al. 1975; Dubos 1928; Ga dne and
Blackwell 1974a; Ga dne and Blackwell 1974b). Howe e , ela i ely li le is known
abou he di e si y and unc ion o o ganisms ha migh pa icipa e in he deg ada ion
o cellulose in soil.
1.2.1. Chemical S uc u e o Cellulose and i s Enzyma ic Hyd olysis
Cellulose is a polysaccha ide composed o β-D-glucose uni s ha a e linked by
β-1,4-glycosidic bonds (Ga dne and Blackwell 1974b; Kuma e al. 2008a). These
linea cellulose chains ha e a a iable deg ee o polyme iza ion o up o 15,000
glucose uni s in co on (Coughlan 1985). Cellulose chains a e linked by
in e molecula hyd ogen bonds and o m he eby c ys alline cellulose (mic o ib ils)
(Figu e 2; O’Sulli an 1997). The deg ee o c ys allini y is a iable (Tee i 1997). The
polysaccha ide cellulose is insoluble and canno be di ec ly assimila ed by celluloly ic
o ganisms (Lynd e al. 2002). Celluloly ic o ganisms exc e e hyd oly ic enzymes, he
so called cellulases, ou side hei cell wall, because hey a e unable o anspo
cellulose ac oss he cell memb ane (Wilson 2008). The igh s uc u e o c ys alline
cellulose is b oken up hese cellulases wha eleases soluble saccha ides (e.g.,
cellodex ins, cellobiose, and glucose) ha a e anspo ed in o he cell and u he
me abolized (Des aux 2005b; Wilson 2008).
Fungal and bac e ial cellulases a e well s udied and ha e been classi ied based
on s uc u al p ope ies and hei speci ic enzyma ic ac i i ies (Hen issa e al. 1998;
Rabino ich e al. 2002). Cellulases a ack c ys alline cellulose in amo phous egions,
i.e., egions ha a e no s ongly s abilized by in amolecula hyd ogen bonds
(Beguin and Aube 1994; Lynd e al. 2002; O’Sulli an 1997). Cellulases may be
eleased as non-complexed (1.2.1.1) o as complexed enzyme sys ems (1.2.1.2)
(Baye and Lamed 1992; Baye e al. 1998b; Des aux 2005b). Non-complexed
cellulases a e p ima ily syn hesized by ae obes, whe eas complexed cellulase
sys ems a e syn hesised by celluloly ic anae obes (e.g., by Clos idium
he mocellum; Baye e al. 1998a; Demain e al. 2005; Wilson 2009a). In gene al, a
igh a achmen o celluloly ic o ganisms o he subs a e is equi ed o e icien ly
hyd olyze cellulose and op imize he consump ion o eleased saccha ides (Beguin
and Aube 1994).
Th ee majo ypes o cellulases a e known (acco ding o Lynd e al. 2002): (i)
endoglucanases (o 1,4-β-D-4-glucan glucanohyd olases; EC 3.2.1.4), (ii)
exoglucanases, including cellobiohyd olases (o 1,4-β-D glucan-4-glucan
cellobiohyd olases; EC 3.2.1.91) and cellodex inases (o 1,4-β-D-glucan
glucanohyd olases; EC 3.2.1.74), and (iii) β-glucosidases (o β-glucoside
glucohyd olases; EC 3.2.1.21). β-glucosidases ac as ex acellula and in acellula
enzymes (Bedino e al. 1985; Inglin e al. 1980; Meye and Cane ascini 1981; Mihoc
and Kluep el 1990). Cellulases a e dis inguished om o he glycoside hyd olases by
hei abili y o (i) hyd olyze β-1,4-glucosidic bonds be ween glucosyl esidues and (ii)
o ac no exclusi ely a chain ends (Baye e al. 1998a; Lynd e al. 2002).
I
NTRODUCTION
4
Figu e 2. Enzyma ic hyd olysis o c ys alline cellulose
. Composi e scheme based
on Baye e al. 1998a; Bisa ia and Ghose 1981; Lynd e al. 2002; Tee i 1997. Enzyme
g oups and he s ep o hyd olysis a which hey a ack he compounds a e indica ed by
numbe s: 1, Endoglucanases (EC 3.2.1.4); 2, Exoglucanases, including cellobiohyd olases
(EC 3.2.1.91) and cellodex inase (EC 3.2.1.74); 3, β-glucosidases (EC 3.2.1.21). Do ed
lines in c ys alline cellulose indica e molecula hyd ogen bonds.
C ys alline cellulose is a acked by endoglucanases (
1
) a in e nal amo phous
egions and andomly cu in o linea cellulose chains o a ious leng hs, leading o
new chain ends (Figu e 2). Exoglucanases (
2
) can also ac on c ys alline cellulose
ib es, p esumably peeling o cellulose chains (Figu e 2). Exoglucanases (
2
) a ack
cellulose chains in a p ocessi e manne on educing and non- educing ends yielding
cellodex ins, cellobiose, and glucose; wi h cellobiose and glucose being he majo
p oduc s (Figu e 2). Soluble cellodex ins and cellobiose a e cu by β-glucosidases
(
3
) in o β-D-glucose (Figu e 2). All ypes o enzymes ac syne gis ically (e.g., Beguin
and Aube 1994; Beguin 1990; Hen issa 1994; Mans ield e al. 1999; Rabino ich e
al. 2002). The saccha ides cellobiose and glucose a e me abolized by a b oad
n
3-6
+
Cellulose (c ys alline)
Cellulose (linea )
β
ββ
β-D-Cellobiose
Cellodex ines β
ββ
β-D-Glucose
Ae obic/Anae obic
Me abolism
1
2
3
2
2
n
3-6
n
3-6
+
Cellulose (c ys alline)
Cellulose (linea )Cellulose (linea )
β
ββ
β-D-Cellobiose
Cellodex ines β
ββ
β-D-Glucose
Ae obic/Anae obic
Me abolism
1
2
3
2
2
I
NTRODUCTION
5
di e si y o ae obic and anae obic celluloly ic and saccha oly ic o ganisms (Baye e
al. 2006; Lynd e al. 2002; Figu e 3 and Figu e 4).
Cellulase enzyme sys ems o bac e ia and ungi unc ion simila and ollow he
same complex induc ion- ep ession mechanisms (Beguin and Aube 1994;
Coughlan 1991; Kuma e al. 2008a; Lynd e al. 2002). Cellulases and
β-glucosidases a e exp essed cons i u i ely a low le els, and excessi e p oduc ion
is egula ed by u he mechanisms (Beguin and Aube 1994; Bus o e al. 1995; Lynd
e al. 2002). In many o ganisms, cellulase biosyn hesis is induced in he p esence o
cellulose o soluble hyd olysis p oduc s like cellobiose o cellodex ins. All known
cellulase sys ems a e addi ionally ep essed in he p esence o low molecula weigh
ca bon compounds such as glucose ha a e mo e easily me abolized han cellulose
(Beguin and Aube 1994; S ewa and Lea he wood 1976). Cellobiose is he main
in e media e o he en i e cellulose hyd olysis p ocess (Co azza e al. 2005). I is no
only an induce o cellulase exp ession, bu also a egula o o endo- and
exoglucanase ac i i y. Bo h ypes o glucanases a e inhibi ed by cellobiose upon a
ce ain concen a ion (e.g., a 0.6 mM cellobiose in cul u es o C. he mocellum;
Beguin and Aube 1994; Be ghem e al. 1975; Gong e al. 1977; Zhang and Lynd
2005). β-glucosidases hyd olyze cellobiose in o wo glucose molecules o p e en
p oduc inhibi ion o glucanases. Hence, he clea age o cellobiose is o en he a e-
limi ing s ep in cellulose hyd olysis (Bha ia e al. 2002; Co azza e al. 2005).
β-glucosidase ac i i y in some celluloly ic ungi is egula ed ia subs a e and p oduc
inhibi ion (e.g. in T ichode ma i idae; Bha ia e al. 2002; Hong e al. 1981). In
con as , he ac i i y o β-glucosidases in he celluloly ic bac e ium C. he mocellum is
no in luenced by glucose (Ka aye a e al. 1992). Se e al o he subs a e- and
enzyme- ela ed ac o s, such as deg ee o subs a e polyme isa ion/c ys allini y o
accessible su ace a ea, a e also de e minan s o enzyma ic cellulose deg ada ion
(Kuma e al. 2008a; Mans ield e al. 1999).
1.2.1.1. Non-Complexed Enzyme Sys ems
Non-complexed cellulase sys ems a e mainly ound in ae obic o ganisms and
a e well s udied in ungi (e.g., T ichode ma sp.; Ilmen e al. 1997; Mandels and
Reese 1957; Yang e al. 2004; Zhang and Lynd 2006) and bac e ia (e.g.,
Cellulomonas sp. and The mobi ida sp.; Chaudha y e al. 1997, I win e al. 1993;
I win e al. 1998; Lamed e al. 1987; Wa en 1996). Each o ganism ha uses non-
complexed enzyme sys ems sec e es a se o indi idual cellulases in o he
en i onmen . The ungus T ichode ma eesei exc e es a se o i e endoglucanases,
wo exoglucanases, and wo β-glucosidases, whe eas species o Cellulomonas
exc e e a leas six endoglucanases and one exoglucanase (Lynd e al. 2002). A
gene al ea u e o mos cellulases is a modula s uc u e including non-ca aly ic
cellulose-binding domains (also e med as ca bohyd a e-binding modules) and
ca aly ic domains (Baye e al. 1998a; Doi 2008; Lynd e al. 2002; Wilson 2009a).
The ca bohyd a e-binding modules media e a achmen o he enzyme o he
cellulose su ace, p esumably o acili a e cellulose hyd olysis by b inging he
ca aly ic domain in close p oximi y o he subs a e, and o ini ia e p ocessing o
exoglucanases (Lynd e al. 2002; Tee i e al. 1998).
I
NTRODUCTION
6
1.2.1.2. Complexed Enzyme Sys ems (Cellulosomes)
Complexed enzyme sys ems a e ypically known om o ganisms li ing in
anae obic en i onmen s (Baye e al. 2004; Lynd e al. 2002). Cellulosomes a e la ge
mul ienzyme complexes wi h nume ous subuni s igh ly linked (Baye and Lamed
1986; Maye e al. 1987). All componen s o m a huge p o ein complex, also called
p o ubozyme (Baye e al. 1994). Cellulosomes a e lexible enough o bind o he cell
wall o he celluloly ic o ganism and simul aneously o he su ace o cellulose. They
media e cellula adhesion and o m a co ido be ween he cell and he subs a e by
changing hei con o ma ion a e binding (Baye e al. 1994; Lynd e al. 2002). The
componen ha dis inguishes cellulosomes om ee non-complex enzyme sys ems
is a non-ca aly ic high molecula weigh sca olding p o ein ha eplaces he
ca bohyd a e-binding module (Baye e al. 1994; Baye e al. 2004; Doi 2008; Wilson
2009a). The cellulosome allows op imal syne gism be ween he exc e ed cellulases
and also minimizes he dis ance o e which hyd olysis p oduc s mus di use. This
acili a es e icien up ake o enzyma ically eleased saccha ides by he o ganism
(Baye e al. 1994; Lynd e al. 2002; Schwa z 2001). Cellulosomes a e well s udied in
anae obic bac e ia (e.g., in Clos idia sp.; Des aux 2005a; Pe i demange e al. 1984;
Schwa z 2001; Zhang and Lynd 2005), bu cellulosome-like s uc u es a e also
known o some anae obic ungi ha a e ound in he diges ion ac o uminan s (Doi
2008; Lynd e al. 2002; O pin 1975; Rabino ich e al. 2002).
1.2.1.3. Ex acellula Cellulose Dis up ing Complex in G am-nega i e Bac e ia
Recen ly, a hi d mechanism o cellulose hyd olysis was p oposed o wo
g am-nega i e bac e ia (Wilson 2008; Wilson 2009b). The ae obe Cy ophaga
hu chinsonii and he anae obe Fib obac e succinogenes nei he use complexed no
non-complexed enzyme sys ems o he deg ada ion o cellulose. Bo h bac e ia do
no encode p ocessi e endo- and exocellulases as a e ypically o o he celluloly ic
o ganisms (Wilson 2008). C. hu chinsonii exclusi ely encodes o non-p ocessi e
endoglucanases and lacks ca bohyd a e-binding modules as well as sca olding
p o eins (Xie e al. 2007). F. succinogenes does also no encode o known
p ocessi e cellulases o sca olding p o eins (Jun e al. 2007; Malbu g e al. 1997).
Howe e , bo h o ganisms can g ow e icien ly on cellulose (Fields e al. 2000;
Nakagawa and Yamasa o 1996). I is sugges ed ha bo h o ganisms use a simila ,
bu no iden ical ex acellula cellulose dis up ing complex ha is bound o he ou e
cell memb ane. This complex emo es linea cellulose chains om c ys alline
cellulose and anspo s hem h ough he ou e memb ane in o he pe iplasmic
space. In he pe iplasm hese chains a e hyd olysed by endoglucanases and
eleased saccha ides a e anspo ed h ough he plasma memb ane in o he cell o
u he me aboliza ion (Wilson 2009a).
1.2.2. Biological Deg ada ion o Cellulose in Soils
Cellulose is a majo sou ce o ca bon o soil mic obial communi ies, and is
deg aded unde oxic and anoxic condi ions (Lynd e al. 2002; Baye e al. 2006).
Decomposi ion o complex plan biomass is ca alyzed by a ious ae obic and
anae obic o ganisms (i.e., mic oo ganisms capable o anae obiosis, which includes
obliga e anae obes and acul a i e ae obes) o he domains Bac e ia and Euka ya
(Bald ian and Valasko a 2008; Lynd e al. 2002). Fo decades, i has been
hypo hesized ha deg ada ion o cellulose is exclusi ely ca ied ou by ungi and
I
NTRODUCTION
7
bac e ia, bu i appea s ha some animals p oduce hei own cellulases (1.3.1), which
di e subs an ially om hose o hei indigenous mic obial communi y (Wa anabe
and Tokuda 2001; Wa anabe and Tokuda 2010).
Celluloly ic and saccha oly ic mic oo ganisms inhabi simila ecological niches,
and compe e o ca bon and ene gy sou ces. Howe e , i was shown ha ae obic
deg ada ion o cellulose by pu e cul u es o Cellulomonas la igena is less e icien
han deg ada ion by mixed cul u es o celluloly ic and saccha oly ic species (Pa el
and Vaughn 1973). Species o Clos idium deg ade cellulose much mo e e icien ly in
he p esence o o he non-celluloly ic bac e ia han in pu e cul u e (Enebo 1949; Ka o
e al. 2004). The saccha oly ic pa ne s imp o e cellulose deg ada ion by e icien
emo al o po en ially inhibi ing hyd olysis p oduc s, e.g. cellobiose and glucose.
These obse a ions indica e ha in e ac ions o axa wi h di e en subs a e spec a
migh be impo an o he e icien deg ada ion o cellulose (1.2.1).
Mo e han 90% o he global amoun o cellulose is deg aded in well ae a ed
ag icul u al, g assland o o es soils (Bas ian e al. 2009; Ku ka 2001; Leschine
1995; Lynd e al. 2002; Vin en e al. 2002). Ae obic ungi and bac e ia a e he main
deg ade s o cellulosic biomass in soil (Bas ian e al. 2009; de Boe e al. 2005), and
hyd olysis o cellulose mainly yields cellobiose and glucose (1.2.1). These
saccha ides a e aken up o ene gy conse a ion and ca bon assimila ion, which
allows o cell g ow h and o ma ion o biomass (Des aux 2006). The main end
p oduc s o cellulose decomposi ion unde oxic condi ions a e ca bon dioxide and
wa e (H
2
O; Baye e al. 2006; Beguin and Aube 1994; Schmid and Ruschmeye
1958).
Anoxic en i onmen s in which anae obic deg ada ion o cellulose occu s a e
mainly ound in he diges i e ac o animals (Leschine 1995; Wa anabe and Tokuda
2001), deep soil sedimen s (Leschine 1995), compos s (de Be oldi e al. 1983), and
wa e -sa u a ed en i onmen s such as eshwa e , ma ine and es ua ine sedimen s,
looded soils, o we lands (Con ad 1996; Glissmann and Con ad 2000; Leschine
1995). Ag icul u al soils (excep o looded ice ields) a e usually wa e -unsa u a ed
and ae a ed. None heless, anoxic mic ozones wi h low concen a ions o oxygen (O
2
)
and low edox po en ial occu (1.6) in which anae obic mic obial ac i i ies can ake
place (Küsel and D ake 1995; Pe -Ridge and Fi es one 2005; Picek e al. 2000;
To sche e al. 2010). Less s udies in es iga ed he deg ada ion o c ys alline
cellulose and plan -de i ed ca bon unde anoxic condi ions in ae a ed soils (Leschine
1995; Lynd e al. 2002), al hough anae obic celluloly ic Bac e ia a e equen ly
isola ed om a ious ypes o ae a ed soil (e.g., An e al. 2005; Ohmiya e al. 2005;
Ozkan e al. 2001; Rampe sad e al. 1998). Ae obic ungi a e also impo an
deg ade s o cellulosic biomass unde oxic condi ions, bu ungal species a e
appa en ly no impo an cellulose deg ade s in soil unde anoxic condi ions (Bald ian
and Valasko a 2008; de Boe e al. 2005). Thus, p oka yo es, i.e., Bac e ia,
ep esen he majo i y o known mic oo ganisms ha a e p ima ily esponsible o he
deg ada ion o cellulose unde anoxic condi ions (Hu and anB uggen 1997)
In anoxic mic ozones, cellulose and cellulose-de i ed saccha ides a e
decomposed by obliga e anae obic and acul a i e ae obic bac e ia ia an
in e media y ecosys em me abolism (D ake e al. 2009; Lynd e al. 2002; Wüs e al.
2009). In pe manen ly lodded o we land soils, e men a ion o soluble saccha ides
by p ima y e men e s (e.g., En e obac e iaceae; Degelmann e al. 2009a) yields a y
acids, alcohols, molecula hyd ogen, and ca bon dioxide
.
These compounds se e as
subs a es o seconda y e men e s, ace ogens, and me hanogens (D ake e al.
2009; D ake e al. 2008; Hambe ge e al. 2008; McIne ney and S uch emeye 2008;
I
NTRODUCTION
14
Celluloly ic o ganisms a ack cellulose wi h hei ex acellula glycosidic enzyme
sys ems, and p oduce cellobiose and glucose (Figu e 4). Mos o he hyd olysis
p oduc s a e di ec ly assimila ed by he celluloly ic mic obe, bu some p oduc s migh
be eleased in o he en i onmen (Baye e al. 1994; Baye e al. 2006). The emo al
o ee cellulose-de i ed suga s by saccha oly ic sa elli e species keeps hese suga s
a low non-inhibi o y concen a ions (Baye e al. 1994; Doi 2008), and hence,
p e en s cellobiose- and glucose-induced inhibi ion o endo- and exocellulases o
celluloly ic o ganisms (1.2.1). P oduc s o saccha ide deg ada ion include a y acids,
alcohols, ca bon dioxide, and molecula hyd ogen (Figu e 4). These compounds a e
u he assimila ed and dissimila ed by o he non-saccha oly ic sa elli e
mic oo ganisms (e.g., ae obic he e o ophs, ni a e educe s, seconda y e men e s,
i on educe s, ace ogens, o me hanogens) o (i) ca bon dioxide unde oxic and (ii)
ca bon dioxide and me hane unde anoxic condi ions (1.2.2) concomi an ly o he
educ ion o al e na i e elec on accep o s such as ni a e (NO
3-
) o e ic i on. In
summa y, he deg ada ion o cellulose in a cellulosic en i onmen is always acili a ed
by a complex mic obial communi y consis ing o celluloly ic and non-celluloly ic
sa elli e o ganisms.
1.6.
En i onmen al and An h opogenic Fac o s In luence
Bac e ial Cellulose Deg ada ion in Ag icul u al Soil
The deg ada ion o cellulose is an impo an communi y unc ion in soil
ecosys ems, and is ca alyzed by anae obic and ae obic saccha ide-deg ading
mic oo ganisms (1.2.2). The bac e ial communi y ha is esponsible o his p ocess
is con on ed wi h changing en i onmen al condi ions, because ag icul u al soil is
no mally a wa e -unsa u a ed, ae a ed, and highly s uc u ed en i onmen ha
comp ises dynamic physicochemical g adien s a biogeochemical in e aces (O e al.
2007; Six e al. 2000; To sche e al. 2010). The dis ibu ion o oxic and anoxic
mic ozones is a iable, spa ially he e ogeneous, and dependen on soil mois u e and
soil agg ega e size (Dasson ille e al. 2004; O e al. 2007; Zausig e al. 1993). Oxic
and anoxic compa men s co-occu on a small scale in he same soil. Ae obic and
anae obic mic oo ganisms exis in hese oxic and anoxic mic ozones, and hei
me abolic ac i i y is de e mined by a ious chemical and physical pa ame e s (B une
e al. 2000; O e al. 2007; To sche e al. 2010; Yada and Malanson 2007).
1.6.1. Changing A ailabili y o O
2
Induces Redox Changes
In wa e -unsa u a ed soils, soil mois u e con en is an impo an soil pa ame e
ha is closely ela ed o he a ailabili y o O
2
and ha can change apidly (Dene e
al. 2001; Mans eld 2004; Vo enhou e al. 2004). The consump ion o O
2
exceeds
he di usion o O
2
a he in e ace o gas- illed and wa e -sa u a ed po es, and
p omo es he o ma ion o anoxic mic ozones (e.g., inside soil agg ega es, Figu e. 3;
B une e al. 2000; Dasson ille e al. 2004; G eenwood 1961). A sudden inc ease o
soil mois u e, e.g. a e a ain e en , can inc ease he size o hese anoxic
mic oen i onmen s (Dene e al. 2001; Mans eld 2004; Vo enhou e al. 2004). O
2
is
he e minal elec on accep o o ae obic espi a ion (B une e al. 2000) and he
deple ion o O
2
acili a es sho e m shi s om oxidizing o educing condi ions
(Dene e al. 2001; Mans eld 2004; Vo enhou e al. 2004). The a ailabili y o O
2
has
a emendous impac on he edox po en ial o soil and also on he ene gy
me abolism/ca abolism o he mic oo ganisms (B une e al. 2000). I is measu ed as
a po en ial di e ence (E
h
in [mV]) be ween an ine elec ode and a e e ence cell
I
NTRODUCTION
15
(Bohn 1971; Fiedle e al. 2007). The sequen ial educ ion o elec on accep o s in
ae a ed soil unde looded condi ions co ela es wi h he he modynamic heo y
(Pe e s and Con ad 1996) ha p edic s ha he educ ion o elec on accep o s
should be sequen ial in he o de o hei s anda d edox po en ials (Zehnde and
S umm 1988). Thus, he edox po en ial o soil is (i) a sui able measu e o he
p esence o oxidizing and educing condi ions, and (ii) o p edic ion o edox
eac ions and mic obial ac i i ies like deni i ica ion, e ic i on educ ion,
e men a ions, and me hanogenesis. A e he deple ion o he p e e ed elec on
accep o O
2
al e na i e elec on accep o s, such as ni a e, manganese (Mn
2+
), o
e ic i on, a e u ilized by anae obic mic oo ganisms, and edox po en ials o soil may
d op o nega i e alues as consequence o he deple ion o oxidized compounds
(Dasson ille e al. 2004; Pe e s and Con ad 1996). The a e a which edox po en ials
d op is dependen in pa on he a ailabili y o o ganic ca bon ha se es as ene gy
and elec on sou ce o mic obial me abolism, and on he concen a ion o al e na i e
elec on accep o s (Bohn 1971; B une e al. 2000; Dasson ille e al. 2004). The
edox po en ial may e u n o mo e oxidizing condi ions due o he inc eased
a ailabili y o O
2
and he ac i a ion o ae obic mic oo ganisms a e a dec ease o soil
mois u e by d ough . Thus, luc ua ion o he a ailabili y o O
2
(as a di ec
consequence o soil mois u e con en ) is an en i onmen al ac o ha impac s on he
ac i i y o mic obial axa in ag icul u al soils (B une e al. 2000; Donnelly e al. 1990;
De e e and Ho wa h 2000; Jacobson and Jacobson 1998; K emen e al. 2005).
Howe e , i is no known, how single membe s o he cellulose-deg ading communi y
espond o luc ua ions in he a ailabili y o O
2
.
1.6.2. Pes icides In luence Biological P ocesses
Mic oo ganisms in ag icul u al soils a e no only challenged by luc ua ing
a ailabili ies o O
2
, bu can also be in luenced by an h opogenic dis u bances (Cox e
al. 1996; Johnsen e al. 2001; Ka ayama and Kuwa suka 1991; Po e and Hayden
2002). The inc eased usage o he bicides, ungicides, and insec icides o e he pas
70 yea s has esul ed in an accumula ion o pes icide esidues in e es ial and
aqua ic en i onmen s (Ake blom 2004; Hille e al. 2008; Tho s ensen e al. 2001).
Pes icides a e subjec ed o mic obial deg ada ion in soils (Chowdhu y e al. 2008;
Gonzalez e al. 2006; Mülle e al. 2001; Sukul and Spi elle 2001), bu hei
deg ada ion is usually slow compa ed o na u al o ganic compounds and occu s
p ima ily unde oxic condi ions (Ha ison e al. 1998; Knaube e al. 2000). Pes icide
deg ada ion is gene ally slowe unde anoxic condi ions (Ha ison e al. 1998;
Knaube e al. 2000). Some pes icides and b eak down p oduc s he eo a e highly
mobile and bioa ailable in soils (Johnsen e al. 2001; Spliid e al. 2006), and hus,
migh impac on soil mic oo ganisms (Chowdhu y e al. 2008; Ge ao e al. 2000;
Ka ayama and Kuwa suka 1991; Ka ayama e al. 1992; Wainw igh 1978). The
he bicide Ben azon inhibi s bac e ial g ow h in pu e cul u e and mic obial ac i i y in
soils (Ce nako a e al. 1991; Galhano e al. 2009; Ma sh e al. 1978). Ben azon-
ea ed soils display dec eased capaci ies o ni ogen mine aliza ion and dini ogen
(N
2
) ixa ion (Galhano e al. 2009; Ma sh e al. 1978). Simila ly, he he bicide MCPA
(4-Chlo o-2-me hylphenoxy ace ic acid) educes g ow h and ac i i y o yeas ,
Pseudomonas pu ida, and Vib io ische i in pu e cul u e (Ah iainen e al. 2003; Cab al
e al. 2003), bu i s oxici y o soil p oka yo es and associa ed p ocesses is
un esol ed. Nonylphenol supp esses cellula espi a o y ac i i y o bac e ia (Hseu
2006, Okai e al. 2000b). The ungicide Chlo o halonil educes cellulose
decomposi ion in ae a ed soils due o inhibi ion o celluloly ic ungi and Bac e ia
I
NTRODUCTION
16
(Ka ayama and Kuwa suka 1991; Ka ayama e al. 1992; Suyama e al. 1993a;
Suyama e al. 1993b). β-glucosidase (1.2.1) ac i i y is inhibi ed by he ungicide
Me alaxyl in soil (Monkiedje and Spi elle 2002; Sukul 2006). Thus, he applica ion o
pes icides migh al e cellulose-dependen ca bon low in soil and migh in luence
associa ed mic obial axa.
1.7.
Hypo heses and Objec i es
The biological deg ada ion o plan -de i ed cellulose is an impo an unc ion o
ag icul u al ecosys ems. The ac i i y o cellulose-u ilizing mic obes in ag icul u al soil
is de e mined by se e al na u al and an h opogenic ac o s, wo impo an ones being
he dis ibu ion o O
2
and he inc easing usage o pes icides. Bo h ac o s migh
in luence he cellulose-deg ading communi y, and migh al e he deg ada ion a es o
cellulose in soil. The p ocess o cellulose deg ada ion has been in ensi ely s udied
(Baye e al. 2006; Be ghem e al. 1975; Hen issa 1994), bu i is widely un esol ed
i he capaci y o soil o deg ade cellulose is also a s able ai o ag icul u al
ecosys ems when he a ailabili y o O
2
changes apidly. Fu he mo e, i is un esol ed
how pes icides a ec ophic in e ac ions in celluloly ic communi ies, and he eby,
in luence cellulose mine aliza ion. The e is s ill a la ge unknown di e si y o cellulose-
deg ading mic oo ganisms, and ew s udies e alua ed he ac i i y and dis ibu ion o
celluloly ic bac e ia in ag icul u al soil (Be na d e al. 2007; Haicha e al. 2007). To
unde s and ca bon low h ough celluloly ic soil communi ies, i is necessa y o
iden i y ac i e mic oo ganisms and o cha ac e ize hei me abolic esponse o
luc ua ions o egula ing en i onmen al ac o s. Thus, he ollowing hypo heses we e
add essed:
Hypo hesis 1: Di e en celluloly ic and saccha oly ic axa ca alyze he
deg ada ion o cellulose in esponse on he p esence o absence o O
2
.
Hypo hesis 2: The selec i e ac i a ion o unc ionally edundan celluloly ic and
saccha oly ic axa yields a s able deg ada ion o cellulose unde luc ua ing
a ailabili ies o O
2
.
Hypo hesis 3: Pes icides impac on he me abolic ac i i y o cellulose-
deg ading mic obial axa and dec ease cellulose deg ada ion.
Objec i es o he doc o al hesis we e o analyze he ae obic and anae obic
dissimila ion and assimila ion o [
13
C]-cellulose, [
13
C]-cellobiose, and [
13
C]-glucose in
oxic and anoxic soil mic ocosms o an ag icul u al soil. Bo h p oka yo ic and
euka yo ic axa ha inco po a ed [
13
C]-labeled ca bon in hei nucleic acids we e
iden i ied by RNA-based s able iso ope p obing (1.4), wi h he p ima y ocus being on
he p oka yo ic communi y. Subsequen ly, axon-speci ic 16S RNA p ime s we e
de eloped o esol e he me abolic esponse o majo celluloly ic and saccha oly ic
bac e ial axa o he in es iga ed soil (i) o luc ua ing a ailabili ies o O
2
using
quan i a i e PCR and (ii) o pes icides du ing he deg ada ion o cellulose and
cellulose-de i ed saccha ides.
M
ATERIAL
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ETHODS
17
2. M
ATERIAL AND
M
ETHODS
2.1. Chemicals, Gases, and Media
Unless o he wise s a ed chemicals and gases we e supplied by Ald ich
(S einheim, Ge many), AppliChem (Da ms ad , Ge many), BioRad (Richmond, USA),
Boeh inge (Mannheim, Ge many), Ca l Ro h (Ka ls uhe, Ge many), Eppendo
(Hambu g, Ge many), Fluka (Buchs, Swi ze land), Me ck (Da ms ad , Ge many),
Sigma (Deisenho en, Ge many), and Rießne (Lich en els, Ge many). Deionised
double-dis illed wa e (ddH
2
O) was p oduced wi h he ul apu e wa e pu i ica ion
sys em Se alpu P o 90 CN (Se al E ich Alhäuse , Ransbach-Baumbach, Ge many).
PCR-H
2
O was p epa ed by il a ion (∅ 0.2 µm) o ddH
2
O and au ocla ing.
RNAse/DNAse- ee wa e (DEPC-H
2
O) was achie ed by ea ing o ddH
2
O wi h
0.1% ( / ) die hylpy oca bona e (DEPC). A e incuba ion (37°C, 1 hou , slow
shaking [300 pm]) he solu ion was au ocla ed (120°C, 1 ba , 20 minu es) o
inac i a ion o DEPC. Di e en cul u e media (Table 2 and Table 3) we e used o
ans o ma ion o compe en cells and cul i a ion o clones. (2.5.12). Composi ion and
p epa a ion as ollows:
Table 2. LB medium (Lu ani-Be ani medium).
Componen Amoun Final Concen a ion
T yp one
10 g 1%
Yeas ex ac
5 g 0.5%
NaCl 5 g 85 mM
ddH
2
O
Ad 1,000 ml -
T yp one, yeas ex ac , and NaCl we e mixed wi h 980 ml ddH
2
O. The pH was
adjus ed o 7.0 and ddH
2
O was added o a inal olume o 1,000 ml. Subsequen ly,
he medium was au ocla ed. Fo he use o LB pla es 1.5% (w/ ) aga was added o
he medium be o e au ocla ing. The medium was pou ed in o s e ile plas ic pe i
dishes and s o ed a 4°C a e solidi ica ion.
Fo blue/whi e-sc eening o clones (2.5.12.3) LB-pla es (LB/Ampicillin/IPTG/X-
Gal-pla es) we e p epa ed ha con ained ampicillin, isop opyl-β-D-
hiogalac opy anoside (IPTG), and 5-B omo-4-chlo o-3-indolyl-β-D-galac opy anoside
(X-Gal). Au ocla ed LB-medium (Table 2) was cooled down o 50°C and 1 ml
ampicillin (100 mg ml
-1
), 1 ml (IPTG, 0.5 M) and 1.6 ml X-Gal (50 mg ml
-1
in N,N’-
dime hyl o mamide) we e added. The medium was pou ed in o s e ile plas ic pe i
dishes and s o ed a 4°C a e solidi ica ion.
M
ATERIAL
A
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ETHODS
18
Table 3. SOC medium (Supe -Op imal-B ow h medium).
Componen Amoun Final Concen a ion
T yp one
2.0 g 2.0%
Yeas ex ac
0.5 g 0.5%
NaCl solu ion (1 M) 1.0 ml 10 mM
KCl solu ion (1 M)
0.25 ml 2.5 mM
Mg
2+
solu ion (2 M)
a
1.0 ml 20 mM
Glucose solu ion (2 M)
1.0 ml 20 mM
ddH
2
O
Ad 100 ml -
a
Mg
2+
solu ion: 24.6 g MgSO
4
, 20.3 g MgCl
2
, ad 100 ml.
T yp one, yeas ex ac , NaCl, and KCl we e mixed wi h 97 ml ddH
2
O and
au ocla ed. A e cool down Mg
2+
and glucose (bo h s e ile il e ed, ∅ 0.2 µm) we e
supplied, and s e ile ddH
2
O was added o inal olume o 100 µl. The medium was
again s e ile il e ed and s o ed a -20°C.
2.2.
Sampling Si e and Soil Cha ac e is ics
Soil samples we e aken om an ag icul u al soil ha was plan ed wi h co n and
loca ed nea he esea ch a m Klos e gu Scheye n, Ge many (Table 4). Soil om
he uppe 20 cm was sampled andomly wi h a spade and samples we e pooled. Soil
ha was no used immedia ely was s o ed a 2°C. Di ec ly be o e use soil was
homogenized by sie ing (mesh size: 2 mm, Re sch, Haan, Ge many).
M
ATERIAL
A
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M
ETHODS
19
Table 4. Soil cha ac e is ics
a
.
Klos e gu Scheye n, Ge many
Coo dina es 48°30.0 ′N, 11°20.7′E
Soil ype
b
Dys ic Cambisol
Tex u e (clay : sil : sand)
c
28 : 33 : 40 (± 0 : 0 : 0) ≙ clay loam (L
2
)
Sampling da es No embe 2007, Ap il 2008/2010, June 2009
W
g
(%) 19.9 ± 3.6
To al C (%)
c
1.2 ± 0.0
To al N (%)
c
0.2 ± 0.0
C/N a io
c
6.9 ± 0.1
pH (in wa e ) 6.5 ± 0.4
pH (in CaCl
2
) 6.0 ± 0.1
I
no ganic ions
d
µmol g
soil DW
-
1
NH
4
+
2.9 ± 4.7
NO
3
-
11.1 ± 8.9
To al Fe
533.8 ± 41.0
To al Mn
17.2 ± 1.5
SO
4
2
-
0.3 ± 0.2
a
Values a e he means o 3 – 21 eplica es wi h s anda d de ia ion.
b
Acco ding o FAO-classi ica ion (Food and Ag icul u e O ganiza ion o he Uni ed Na ions;
Fuka e al. 2008).
c
Depa men o Soil Ecology, Uni e si y o Bay eu h, Ge many. L
2
(Ge man soil
classi ica ion; Eckelmann 2005).
d
Ins i u e o Cen al Analy ics, Uni e si y o Bay eu h, Ge many.
W
g
, G a ime ic wa e con en .
DW, D y weigh .
2.3.
Soil Mic ocosms
Mic ocosms we e p epa ed as slu ies (soil : wa e , 1 : 2.5) o by using ield
esh soil wi hou addi ional wa e . Values o subs a e- ee con ols we e included in
any expe imen o de e mine indigenous ca bon u no e and linked p ocesses.
Subs a e- ee con ols we e used o co ec ion o subs a e-supplemen ed
ea men s o di ec ly show subs a e- ela ed p ocesses. Values ob ained in
subs a e- ee con ols we e sub ac ed om alues in subs a e-supplemen ed
ea men s a he co esponding ime poin . Incuba ions we e pe o med a 15°C o
20°C in he da k.
M
ATERIAL
A
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M
ETHODS
20
2.3.1. T ea men s o S able Iso ope P obing (SIP)
S able iso ope p obing expe imen s we e pe o med o esol e ae obic and
anae obic dissimila ion o h ee model subs a es, and o iden i y p oka yo es ha
assimila ed subs a e-de i ed ca bon (2.5.6). C ys alline cellulose, he disaccha ide
cellobiose, and he monosaccha ide glucose we e used in ei he i s [
12
C]- o [
13
C]-
iso opic o m (uni o mly labeled; IsoLi e, Wageningen, The Ne he lands).
Field esh, sie ed soil was mixed wi h 2.5 olumes o s e ile oxic o anoxic
double dis illed wa e (ddH
2
O) and mixed by ex ensi e manual shaking o 5 minu es.
Slu ies we e placed on ice and lushed wi h s e ile dini ogen (100%; Rießne ,
Lich en els, Ge many) o s e ile ai o 1 hou . Slu ies we e homogenized on an end-
o e -end shake (Reax 2, Heidolph, Schwabach, Ge many) o 1.5 hou s a 5°C,
di ided in o 80 ml aliquo s, and illed in bu yl ubbe s oppe ed 500 ml (anoxic) o 1 l
(oxic) lasks (Mülle & K empel, Bülach, Swi ze land) wi h dini ogen o ai as
a mosphe e. A e he addi ion o subs a e (2.3.1.1., 2.3.1.2) incuba ion was
pe o med a 15°C on an end-o e -end shake o 84 days (cellulose) o 24 days
(cellobiose, glucose). E e y 7 days cellulose-supplemen ed ea men s we e lushed
o 15 minu es wi h s e ile dini ogen o ai . Cellobiose- o glucose-supplemen ed
ea men s we e lushed e e y 2 days (oxic) o 4 (anoxic) days. Exchange o he
headspace was pe o med o minimize ‘c oss-labeling’ by ei he he au o ophic o
he e o ophic ixa ion o he [
13
C]-ca bon dioxide o med du ing he deg ada ion o he
p ima y labeled subs a e.
2.3.1.1. Supplemen a ion o Cellulose
0.2 g o [
13
C]-cellulose o [
12
C]-cellulose was added once due o he insolubili y
o his polyme . The amoun o supplemen ed cellulose co esponds o app oxima ely
6.6 mmol o ca bon based on he assump ion ha 1 mol cellulose (independen o
deg ee o polyme iza ion) is equi alen o 1 mol glucose (MW = 180 g mol
-1
). [
13
C]-
ea men s we e duplica ed, [
12
C]- ea men s we e no eplica ed. Endogenous
ca bon u no e was moni o ed in unsupplemen ed oxic and anoxic con ols (2.3).
2 ml o he liquid and 0.2 ml o he gaseous phase we e aken e e y 7 days wi h
s e ile sy inges o analysis o me hane, ca bon dioxide and molecula hyd ogen
(2.4.6), soluble o ganic compounds (2.4.7), ino ganic compounds (2.4.10), and pH
measu emen s (2.4.4). Gases we e immedia ely analysed a e sampling, ollowed by
he exchange o he headspace. 2 ml o he liquid phase we e addi ionally aken o
molecula analyses a day 0, 35, and 70 (2.5), and s o ed a -80°C un il use. In
collabo a ion wi h D . An onis Cha zino as (Depa men o Ecological Mic obiology,
Helmhol z Cen e o En i onmen al Resea ch, UFZ Leipzig, Ge many) cDNA (2.5.7)
samples o day 0, 35 and 70 de i ed om oxic and anoxic [
12
C]- and [
13
C]-g adien s
we e sen o UFZ Leipzig and used o de e mine i also euka yo ic o ganisms
(i.e., ungi and p o is s) we e labeled in he expe imen .
M
ATERIAL
A
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M
ETHODS
21
2.3.1.2. Supplemen a ion o Cellobiose and Glucose
The subs a e, i.e., [
12
C]- o [
13
C]-cellobiose o [
12
C]- o [
13
C]-glucose, was
pulsed pe iodically, i.e., daily in oxic ea men s and e e y wo days in anoxic
ea men s. In o al, 0.5 ml o a subs a e s ock solu ion (40 mM) we e added 12
imes. The concen a ion o glucose and cellobiose (0.25 mM pe pulse) was i e
imes g ea e han maximum concen a ion de ec ed in soils (Hill e al. 2008;
Medei os e al. 2006), and hus was supplied a amoun s ha ep esen ed a
comp omise be ween he low in si u concen a ions and ha needed o e ec i e
[
13
C] inco po a ion in he RNA pool. [
13
C]-supplemen ed ea men s we e se up in
iplica es, [
12
C]-supplemen ed and unsupplemen ed con ol ea men s (2.3) in
duplica es. The o al amoun s o cellobiose and glucose pulsed we e 0.24 mmol,
which co esponds o 2.88 and 1.44 mmol o ca bon, espec i ely.
Analogous o cellulose-supplemen ed ea men s (2.3.1.1) me hane, ca bon
dioxide and molecula hyd ogen
(2.4.6), soluble o ganic compounds (2.4.7), ino ganic
compounds (2.4.10), pH measu emen s (2.4.4), and exchange o he headspace
we e pe o med e e y 2 days (oxic) o 4 days (anoxic). Addi ionally, 2 ml o he liquid
phase we e sampled o molecula analyses ha we e pe o med a day 0, 6, and 12
in oxic ea men s, and a day 0, 4, 20, and 24 in anoxic ea men s (2.5).
2.3.2. T ea men s in Sel -Cons uc ed Incuba ion Chambe s
The e ec o pe iodically changing O
2
a ailabili ies on he deg ada ion o
ca boxyme hyl-cellulose (CMC) and cellobiose was analysed in sel -cons uc ed
incuba ion chambe s (Figu e 5). P e iously iden i ied celluloly ic and saccha oly ic
axa (2.5.6) we e analysed o hei me abolic esponse on he a ailabili y o O
2
.
2.3.2.1. Design o Incuba ion Chambe s
Incuba ion chambe s (Figu e 5) we e cons uc ed in close collabo a ion wi h G.
Kü ne , and P. Schmid and colleagues (Wissenscha liche We ks ä en, Uni e si y o
Bay eu h, Ge many). The cen al elemen was a plexiglas cylinde (inne ∅: 100 mm;
heigh : 125 mm; wall hickness: 10 mm) ha was s uck o a plas ic disk (150 mm x
150 mm). The co e pla e (∅: 150 mm) was equipped wi h a closable senso po o
inse ion o pH/ edox senso s, wo ball al es o gas in- and gas ou le , and an
exchangeable sep um (∅: 10 mm, hickness: 3 mm) o sampling o he liquid phase.
The co e pla e was ixed gas igh wi h ou h ead- o ming sc ews. The gas inle
al e was linked by a plexiglas ube wi h he gas dis ibu ion pla e loca ed a he
bo om o he chambe s. Syn he ic ai o dini ogen was supplied ia he gas inle o
ae a e he slu ies om below. Gas was eleased om he gas ou le .
M
ATERIAL
A
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M
ETHODS
22
Figu e 5. Incuba ion chambe .
Senso po was used o pH and edox po en ial
measu emen s. S e ile syn he ic ai o dini ogen we e supplied a a gas inle , and slu ies
we e ae a ed om below by a gas dis ibu ion pla e. Liquid samples we e aken a he
sep um wi h a s e ile sy inge. Gas was eleased om he gas ou le . Val es o gas inle and
ou le we e closed du ing anoxic incuba ion, bu a he oxic phases syn he ic ai was
pe manen ly lushed h ough he soil slu y.
2.3.2.2. Incuba ion Condi ions
50 g o ield esh, sie ed soil was illed in a chambe , 250 ml s e ile oxic o
anoxic ddH
2
O was added, chambe s we e closed, and he slu ies we e mixed
ho oughly by manual shaking. The chambe s we e di ided in o ou g oups: anoxic
con ols (pe manen ly anoxic, no subs a e supplemen a ion), edox con ols
(changing O
2
s a us, no subs a e supplemen a ion), cellobiose-supplemen ed
ea men s (changing O
2
s a us), and ca boxyme hyl-cellulose (CMC)-supplemen ed
ea men s (changing O
2
s a us). Chambe s we e hen lushed wi h s e ile dini ogen
(100% Rießne , Ge many, 3 eplica es) o wi h syn he ic ai (80% dini ogen, 20% O
2
,
Rießne , Ge many; 9 eplica es) o 1 hou . Cellobiose and CMC we e pe iodically
supplemen ed (0.5 mM subs a e pe pulse), i.e., he subs a e was supplemen ed
a e 48, 96, and 168 hou s o incuba ion. To al amoun s o cellobiose and CMC
pulsed we e 0.6 mmol, which co esponds o 7.2 mmol and 4.8 mmol o ca bon,
espec i ely. A e 48 hou s he chambe s we e lushed wi h dini ogen o 1.5 hou s
o achie e anoxic condi ions in ea men s o me ly incuba ed unde O
2
con aining
a mosphe e. Re-ae a ion wi h O
2
was pe o med a e 168 hou s in edox con ols
and subs a e-supplemen ed ea men s by cons an lushing wi h syn he ic ai un il
he end o incuba ion. Du a ion o he anoxic incuba ion was used since simila
pe iods o O
2
deple ion a e likely o occu in upland soils (Pe -Ridge and Fi es one
2005). T ea men s we e se up in iplica es and chambe s we e incuba ed a 20°C. 2
Senso po wi h edox elec ode
Gas ou le
Plexiglas ube
Gas dis ibu ion pla e
Sep um
Gas inle
M
ATERIAL
A
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M
ETHODS
23
ml o he liquid phase we e aken o de e mina ion o soluble o ganic compounds
(2.4.7). Addi ionally 2 ml liquid sample we e aken a e 0, 48, 102, 168, and 196
hou s o molecula analyses (2.5), i.e., quan i ica ion o he ansc ip numbe s o he
ollowing domain- and amily-le el axa (2.5.10): A chaea
To al
, Bac e ia
To al
,
Mic ococcaceae/Cellulomonadaceae (phylum Ac inobac e ia), Kineospo iaceae/
Noca dioidaceae (phylum Ac inobac e ia), Clus e I Clos idiaceae (phylum
Fi micu es), Planc omyce aceae (phylum Planc omyce es), he new axa ‘Cellu1-3’
(phylum Bac e oide es), ‘Sphingo1-4’ (phylum Bac e oide es), and ‘Deha1’ (phylum
Chlo o lexi).
2.3.3. T ea men s o Resol e Impac o Pes icides on he Deg ada ion o
Saccha ides
Oxic and anoxic soil ea men s we e se up o analyse he impac o pes icides
on he ae obic and anae obic deg ada ion o cellulose and cellobiose, and he
in ol ed p oka yo ic soil communi y. P oka yo ic axa ha we e p e iously iden i ied
o be pa o he ac i e celluloly ic and saccha oly ic soil communi y (2.5.6) we e
quan i ied by 16S RNA gene based quan i a i e PCR (qPCR; 2.5.10). Fi e
pes icides we e es ed o po en ial inhibi o y e ec s in sho - e m p eincuba ions
(2.3.3.1). The lack o an aqueous phase wi h de ined olume in cellulose-
supplemen ed soil ea men s p ohibi s exp ession o alues in mM. The e o e, in all
ea men s de e mining pes icide impac any concen a ion is gi en in µmol g
soil
DW-1
.
2.3.3.1. Pes icides
Fi e xenobio ic compounds (Table 5) we e es ed o hei po en ial o impac on
he deg ada ion o he model subs a e cellobiose. Compounds comp ise ing edien s
o comme cially a ailable pes icide o mula ions. The e m ‘pes icide’ is used o
deno e hese compounds h oughou he wo k.
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n
gas
, amoun o gas in gaseous phase (µmol); V
gas
, olume o gas phase (ml); c, measu ed
gas concen a ion (ppm); V
ak ,mol
, mola olume o gas unde ac ual condi ions (ml); p
ak
,
ac ual ai p essu e (mba ); p
ü
, o e p essu e in incuba ion lasks (mba ).
The mola gas olume a ac ual condi ions (V
a k,mol
) was de e mined based on
he ideal gas law (Equa ion 4).
Equa ion 4. Ideal gas law.
ak
ak
molak
pT
TVp
V•
•
•
=
1
11
,
p
1
, s anda dized ai p essu e (mba ); V
1
, s anda dized mola gas olume (ml); T
1
,
s anda dized empe a u e (K); T
ak
, ac ual empe a u e (K).
The amoun o gas physically and chemically dissol ed in he liquid phase is
based on Equa ion 5 and Equa ion 6. Bunsen solubili y coe icien (α) is a iable and
is dependen on he gas ype and he ac ual empe a u e (Blachnik 1998; Table 8).
Equa ion 5. Amoun o physically dissol ed gas.
ak
üak
molak
lpgel
p
pp
Vc
Vn
+
•••=
α
,
,
V
l
, olume o liquid phase (ml);
Equa ion 6. Amoun o chemically dissol ed gas.
pHpk
pgelcgel
a
nn
+−
•= 10
,,
Table 8. Bunsen solubili y coe icien s o ca bon dioxide, me hane, and
molecula hyd ogen a di e en empe a u es (Blachnik 1998).
Bunsen solubili y coe icien α
α α
α (in wa e )
293.15 K (20°C) 298.15 K (25°C)
CO
2
0.850 0.740
CH
4
0.032 0.029
H
2
0.018 0.015
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2.4.7. Soluble O ganic Compounds
Concen a ion o alcohols, a y acids, and suga s was measu ed by high
pe o mance liquid ch oma og aphy (HPLC; Eh lich e al. 1981; Ma hies e al. 1993)
in liquid samples o mic ocosm expe imen s (2.3). Soluble o ganic compounds in
cellulose-supplemen ed ea men s (2.3.3.3) we e de e mined a e mixing soil wi h
2.5 olumes o ddH
2
O and homogeniza ion on an end-o e -end shake o 10
minu es a 5°C. Liquid samples we e cen i uged (1-15K Sa o ius mic ocen i uge,
Sigma Labo zen i ugen, Os e ode am Ha z, Ge many; 13,000 x g, 10 minu es, 4°C)
and supe na an was mic o il a ed (HPLC nylon il e , po e olume 0.2 µm,
In och oma, Zug, Swi ze land) in o lange bo les wi h aluminium caps (VWR
In e na ional, Da ms ad , Ge many). Samples we e di ec ly analysed a e il a ion in
a Hewle Packa d 1090 Se ies II HPLC sys em (Palo Al o, USA) equipped wi h an
au osample (Table 9). Injec ion olume was 20 µl. Ex e nal s anda ds wi h known
concen a ions o soluble o ganic compounds we e used o quan i ica ion.
Table 9. Pa ame e s o HPLC sys em o soluble o ganic compounds.
Hewle Packa d 1090 Se ies II
De ec able compounds Alcohols, a y acids, suga s
P ecolumn Ca bo-H 4 x 3 mm ID
Column Aminex Ion Exclusion HPX-87H; 300 x 7.8 mm
(BioRad, Richmond, USA)
De ec o s G1362A RID, G1314B VWD (210 nm)
(Se ies 1200, Agilen Technologies, Böblingen, Ge many)
Mobile phase H
3
PO
4
(4 mM)
O en emp. [°C] 60
Flow a e [ml min
-
1
] 0.8
So wa e ChemS a ion (Agilen Technologies, Böblingen, Ge many)
emp., Tempe a u e.
RID, Re ac i e index de ec o .
VWD, Va iable wa eleng h de ec o .
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2.4.8. Cellulose
Deg ada ion o cellulose (2.3.3.3) was de e mined by measu ing cellulose
weigh loss o bu ied cellulosic pape shee s (Munie -Lamy and Bo de 2000). This
p ocedu e is a sui able me hod o measu e deg ada ion o c ys alline cellulose (Hi oki
and Wa anabe 1996; Jacobson and Jacobson 1998; Ku ka 2001; Munie -Lamy and
Bo de 2000). Shee s we e d ied a 40°C o 4 hou s in a d ying cabine (The mo
Fishe Scien i ic, Wal ham, USA) and weighed on a MC1 analy ic AC 120 S balance
(AS Wäge echnik, Ga ding, Ge many) be o e inse ion in o he soil. Shee s we e
gen ly emo ed wi h o ceps a e incuba ion. Shee s we e washed wi h s e ile ddH
2
O
o emo e a ached soil pa icles, d ied, and weighed. The di e ence in cellulose
weigh loss was used o calcula ion o he po en ial inhibi ion by pes icides (2.3.3.4).
2.4.9. Pes icides
Pes icides we e ex ac ed and quan i ied in pes icide-supplemen ed ea men s
(2.3.3.3; Liu e al. 2011). The pes icide ex ac ion p ocedu e was op imized and he
ex ac ion e iciencies we e 102.4 ± 4.1% o Ben azon, 98.3 ± 2.8% o MCPA, and
92.1 ± 2.5% o Nonylphenol. Ben azon and Nonylphenol we e ex ac ed wi h 70%
e hanol, and MCPA wi h 10 mM NaOH (in iplica es). One olume o soil was mixed
wi h h ee olumes o sol en . Mix u es we e incuba ed o 2 hou s a 15°C on an
end-o e -end shake and cen i uged (13,000 pm, 10 minu es, oom empe a u e).
Samples we e mic o il e ed and injec ed in a Agilen Technologies 1200 Se ies HPLC
ins umen (Agilen Technologies, Böblingen, Ge many) ha was equipped wi h a
1200 Se ies diode-a ay de ec o (Agilen 1200 se ies, Agilen Technologies,
Böblingen, Ge many) (Table 10). Pes icides we e sepa a ed wi h on a Mul oHigh 100
RP 18-5µ column (250 x 4 mm) wi h a p e-column (20 x 4 mm; CS Ch oma og aphie,
Lange wehe, Ge many) a cons an empe a u e (30°C). Peak pu i y was con i med
by online spec a om 220 o 340 nm. Ex e nal s anda ds wi h known concen a ions
o pes icide we e used o calib a ion. Signals we e eco ded a 230 nm wi h he
so wa e ChemS a ion (Ve sion B.04.01; Agilen Technologies, Böblingen, Ge many)
Table 10. Pa ame e s o pes icide measu emen s by HPLC.
Ben azon Nonylphenol MCPA
Injec ion emp. [°C] 30 30 30
De ec o emp. [°C] 30 30 30
Mobile phase 50:50 ( / %)
Ace oni ile :
C
2
H
3
NaO
2
(20 mM)
70:30 ( / %)
Ace oni ile :
ddH
2
O
50:50 ( / %)
Ace oni ile :
C
2
H
3
NaO
2
(20 mM)
Flow a e [ml min
-
1
] 1.0 2.5 1.0
Re en ion ime [min] 7 - 9 11 - 12 7 - 9
Re e ence This wo k This wo k Liu e al. 2011
emp., Tempe a u e.
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2.4.10. Ino ganic Compounds
2.4.10.1.
Ni a e (NO
3-
) and To al Amoun s o Ammonium (NH
4+
), I on,
Manganese, and Sulpha e
Compounds we e analysed by s anda dized colo ime ic me hods by co-
wo ke s o he Ins i u e o Cen al Analy ics, Uni e si y o Bay eu h. Ex e nal
s anda ds wi h known concen a ions we e used o quan i ica ion.
2.4.10.2. Fe ous I on (Fe
2+
)
Concen a ions o e ous i on we e de e mined pho ome ically (Tamu a e al.
1974). 20 µl sample we e mixed wi h 980 µl HCl (0.5 N), incuba ed o 1 hou a oom
empe a u e, and mic o il a ed o emo e soil pa icles. 450 µl sample we e mixed
wi h 50 µl ace a e bu e (200 g CH
3
COONH
4
, 250 ml CH
3
COOH, ad 500 ml ddH
2
O)
and 50 µl o a phenan h oline solu ion (1 g 1,10-Phenan h olinium-chlo ide-
monohyd a e ad 200 ml ddH
2
O), leading o he de elopmen o a ed-colou ed Fe-
phenan h oline-complex. 160 µl o he mix u e we e immedia ely ans e ed o
mic o i e pla es (O ange Scien i ic, B aine-l’Alleud, Belgium) and ex inc ion was
measu ed a 512 nm by a µQuan mic opla e spec opho ome e (BioTek, Bad
F ied ichshall, Ge many). The so wa e Gen5 (Ve sion 1.04.5; BioTek, Bad
F ied ichshall, Ge many) and ex e nal s anda ds we e used o quan i ica ion.
2.5. Molecula Me hods
2.5.1. Ex ac ion o Nucleic Acids
Nucleic acids, i.e., DNA and RNA, we e co-ex ac ed acco ding o a modi ied
p o ocol o G i i hs e al. 2000. All s eps we e pe o med in ce i ied RNase-and
DNase- ee ubes. Fil e ed ips we e used o a oid con amina ion o nucleic acids
wi h hyd oly ic enzymes. Cen i uga ion was ca ied ou on an analy ical cen i uge
(1-15K Sa o ius mic ocen i uge, Sigma, Os e ode am Ha z, Ge many) a 13,000 x g
o 5 minu es a 4°C unless o he wise s a ed. 2 ml soil slu y we e ans e ed o 2 ml
sc ew-capped ubes (VWR In e na ional, Da ms ad , Ge many), cen i uged, and
supe na an was disca ded. 1 g o baked (12h, 200°C) zi conium beads (0.5 g ∅ 0.1
mm, 0.5 g ∅ 0.5 mm; Ca lRo h, Ka ls uhe, Ge many), 500 µl p ehea ed ex ac ion
bu e (60°C, 5% CTAB, 350 mM NaCl, 120 mM po assium phospha e bu e
[con aining KH
2
PO
4
and K
2
HPO
4
], pH 8), and 500 µl
phenol/chlo o o m/isoamylalcohol (25:24:1, pH 8) we e added. Samples we e bead
bea en wo imes a 5.5. m/s in a Bead Bea e (Fas P ep FP 120, The mo Sa an ,
USA) o 30 seconds and cen i uged. Supe na an was ans e ed o s e ile 1.5 ml
ubes, 500 µl chlo o o m/isoamylalcohol (24:1) we e added, samples we e mixed,
and cen i uged. Supe na an was ans e ed o s e ile 2 ml ubes and 2 olumes o
p ecipi a ion bu e (30% PEG6000, 0.1 M HEPES, pH 7) we e added un il he
solu ion was clea . Nucleic acids we e p ecipi a ed a oom empe a u e (20 – 25°C)
o 2 hou s and cen i uged o pelle nucleic acids. The pelle was washed wi h 500 µl
ice cold e hanol (70%, -20°C) o dissol e co-p ecipi a ed sal s, cen i uged, and he
supe na an was disca ded. Nucleic acids we e d ied a oom empe a u e and
dissol ed in 52 µl DNase/RNase- ee ddH
2
O. Nucleic acid ex ac ion was checked
spec opho ome ically (2.5.3.1) and ex ac s we e s o ed a -80°C.
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2.5.2. Sepa a ion o DNA and RNA
2.5.2.1. Solid Phase Columns
Co-ex ac ed DNA and RNA (2.5.1) we e sepa a ed wi h he QIAGEN
RNA/DNA Mini Ki (QIAGEN, Hilden, Ge many) acco ding o he manu ac u e ’s
p o ocol. The p ocedu e was s a ed a s ep 3 o he p o ocol o ‘Isola ion o o al
RNA and genomic DNA om Bac e ia’, a p o ocol sui able o o al RNA amoun s
smalle han 40 µg. A e sepa a ion DNA was dissol ed in 50 µl, RNA in 30 µl
DNase/RNase- ee ddH
2
O. DNA and RNA we e quan i ied (2.5.4.2) and samples
we e s o ed a -80°C.
2.5.2.2. Enzyma ic Sepa a ion
An al e na i e enzyme-based me hod was applied o sepa a e nucleic acids.
RNA was ob ained by diges ion o DNA wi h DNase I (1U µl
-1
; Fe men as, S . Leon-
Ro h, Ge many). 3 µl eac ion bu e (10x) and 1 µl DNAse I we e mixed wi h 30 µl
co-ex ac and samples we e incuba ed on a he momix com o (Eppendo ,
Hambu g, Ge many) a 37°C o 45 minu es unde cons an shaking (300 pm). DNA
was ob ained by diges ion o RNA wi h RNase A (10 µg µl
-1
; Fe men as, S . Leon-
Ro h, Ge many). 1 µl RNase A we e added o 20 µl co-ex ac and samples we e
incuba ed o 20 minu es a oom empe a u e (20 – 25°C). Enzyma ic diges ions
we e s opped by pu i ica ion o nucleic acids by isop opanol/Na-ace a e p ecipi a ion
(2.5.3.1).
2.5.3. Pu i ica ion and P ecipi a ion o Nucleic Acids
2.5.3.1.
Isop opanol/Sodium Chlo ide P ecipi a ion
Nucleic acids ha we e subjec ed o DNase I o RNase A diges ion (2.5.2.2)
we e pu i ied by isop opanol p ecipi a ion (Samb ook and Russell 2001). 0.7 olumes
o ice cold isop opanol (100%, -20°C) and 0.1 olum e o NaCl (5 mM) we e added o
1 olume o nucleic acids. Samples we e incuba ed o a leas 12 hou s a -20°C,
and nucleic acids we e p ecipi a ed by cen i uga ion (18,000 x g, 45 minu es, 15°C).
The DNA o RNA pelle was washed wi h 500 µl ice cold e hanol (70%, -20°C), d ied
a oom empe a u e, and dissol ed in 22 µl DNAse/RNAse- ee ddH
2
O. 2 µl o
nucleic acid solu ion we e used o quan i ica ion o DNA o RNA by luo escence
based me hods (2.5.4.2). DNA and RNA samples we e s o ed a -80°C.
2.5.3.2. Gel Ex ac ion
PCR p oduc s ampli ied o RFLP analyses (2.5.11) and PCR p oduc s o he
cons uc ion o gene lib a ies (2.5.12) we e pu i ied by gel ex ac ion (Mon age Gel
Ex ac ion Ki , Millipo e, Bed o d, USA) a e he manu ac u e ’s p o ocol. Samples
we e loaded on a 1% aga ose gel ha was p epa ed wi h modi ied TAE bu e
(40 mM T is-Ace a e, 0,1 mM Na
2
EDTA, pH 8,0; Millipo e, Bed o d, USA). Aga ose
gel elec opho eses was pe o med o 1 hou a 70 mV as desc ibed (2.5.5). Bands
we e excised unde UV-ligh wi h a s e ile kni e and loaded on Mon age DNA Gel
Ex ac ion De ice.
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2.5.3.3. Fil e Pla es
PCR p oduc s ha we e subjec ed o mung bean endonuclease diges ion
(2.5.11.1) and PCR p oduc s ep esen ing clone inse sequences o e-ampli ied
16S RNA genes he eo (2.5.9.3, 2.5.10.2) we e pu i ied wi h Millipo e PCR
96
Cleanup Pla es (Millipo e Coope a ion, Bed o d, USA) a e manu ac u e ’s p o ocol.
Samples we e loaded on o he il e s and pla es we e e acua ed o 12 minu es wi h
a suc ion pump (KNF Neube ge , Bal e swil, Swi ze land). PCR p oduc s we e
dissol ed in 25 µl is bu e (10 mM, pH 8.5) and ans e ed in o s e ile 0.5 ml ubes
(LoBind ubes, Eppendo , Hambu g, Ge many) by pipe ing.
2.5.4. Quali y Con ol and Quan i ica ion o Nucleic Acids
2.5.4.1. Spec opho ome y
Quali y o nucleic acid ex ac ion was de e mined wi h a Nanod op ND-1000
spec opho ome e (PEQLAB Bio echnology GmbH, E langen, Ge many) and
supplied so wa e. Abso p ion a 260 nm, 280 nm, and 230 nm was measu ed in 2 µl
sample. One abso p ion uni a 260 nm (A
260
) co esponds o a concen a ion o
50 µg
DNA
ml
-1
and 33 µg
RNA
ml
-1
, espec i ely (Lo speich and Engels 2006;
Samb ook and Russell 2001). The a enua ion coe icien A
260
/A
280
gi es in o ma ion
abou possible con amina ions o nucleic acids wi h phenol, p o eins, o humic acids.
An ideal coe icien lies be ween 1.6 and 2.0 (Samb ook and Russell 2001).
Addi ionally he abso p ion spec um should be low a 230 nm, because humic acids
exhibi a s ong abso p ion a his wa eleng h (Tsu suki and Kuwa suka 1979). All
DNA and RNA samples o he cu en s udy showed a good quali y and we e no
con amina ed wi h humic acids.
2.5.4.2. Pico-/RiboG een-Based Quan i ica ion
DNA and RNA samples o nucleic acids a e ex ac ion (2.5.2), pu i ied PCR
p oduc s (2.5.3), and PCR p oduc s ha we e subjec ed o es ic ion diges ion o
RFLP analysis (2.5.11.2) we e quan i ied wi h a luo escence-based me hod
(Samb ook and Russell 2001). Fluo escence-based me hods a e insensi i e o
in e e ences caused by p o eins o o he con amina ions, and allow exac
quan i ica ion o e y low amoun s o nucleic acids (minimum: 25 pg
DNA
ml
-1
[Jones e
al. 1998] o 20 ng
RNA
ml
-1
[Laba ca and Paigen 1980]). Quan i ica ion o DNA and
RNA was pe o med in mic o i e pla es wi h he Quan -iT-PicoG een dsDNA eagen
Ki and he Quan -iT-RiboG een Quan i a ion Reagen Ki (bo h In i ogen, Ka ls uhe,
Ge many), espec i ely. A FLx800 mic opla e luo ome e (BioTek, Bad
F ied ichshall, Ge many) was used and quan i ica ion was pe o med acco ding o
he manu ac u e ’s p o ocol. Fo quan i ica ion o RNA an assay sui able o 1 – 50 ng
ml
-1
was used (i.e., ‘low ange’). Ex e nal s anda ds wi h known concen a ions o
DNA o RNA we e en ained in e e y measu emen . Flo escence signals we e
eco ded wi h he so wa e Gen5 (BioTek, Bad F ied ichshall, Ge many).
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2.5.5. Aga ose Gel Elec opho esis
PCR p oduc s we e quali ied wi h ho izon al aga ose gel elec opho esis, a
me hod ha sepa a es nucleic acids in an elec ical ield dependen on hei size (Aaij
and Bo s 1972; Samb ook and Russell 2001). Aga ose gels (1%) we e p epa ed
wi h 1x TAE bu e (40 mM T is-HCl, 20 mM CH
3
COOH, 1 mM EDTA, pH 8). The
bu e ed mix u e was hea ed in a MWG 756 E mic owa e (Cla onic, Kempen,
Ge many) un il aga ose was ully dissol ed. A e cool down o 60°C 0.05 mg ml
-1
e hidium b omide (3,8-Diamino-5-e hyl-6-phenyl-phenan h idiumb omid) we e added
o isualiza ion. E hidium b omide in e cala es wi h DNA and shows a s ong
luo escence unde UV-ligh a e binding o DNA. Liquid gels we e illed in a gel ack,
ans e ed o a mig a ion chambe (Techne, Jahnsdo , Ge many) a e solidi ica ion,
and co e ed wi h unning bu e (1x TAE). Samples we e p epa ed by mixing 5 µl
sample wi h 1 µl 6 x Blue O ange loading dye (P omega, Madison, USA) o sel
p epa ed 6 x loading dye (0.05% b omophenol blue, 0.05% xylene cyanol, 55%
glyce ol). Samples we e illed in o he gel slo s. 2 µl o a DNA ladde (MWM-1, 200 –
10,000 bp, Bila ec, Vie nheim, Ge many) we e used o de e mina ion o p oduc
leng h. Elec opho esis was pe o med a 120 V o 20 – 30 minu es. Visualiza ion o
bands by UV-ligh (302 nm) was ca ied ou on a ansillumina o (UVT-20M, He olab,
Wiesloch, Ge many) and a pho og aphically based documen a ion sys em (Canon
Powe Sho G5, Canon, K e eld, Ge many; so wa e: Remo e Cap u e).
2.5.6. RNA S able Iso ope P obing (RNA SIP)
RNA-based s able iso ope p obing is a cul u e-independen me hod o
iden i ica ion o mic oo ganisms ha a e esponsible o a ce ain p ocess (e.g.,
cellulose deg ada ion; Mane ield e al. 2002b). In his s udy, he p o ocol o Whi eley
e al. 2007 was used in a sligh ly modi ied o m. S able iso ope p obing expe imen s
we e pe o med in oxic and anoxic ea men s (2.3.1)
[
13
C]-labeled subs a es a e added o he en i onmen al sample and incuba ed
(Figu e 6). Mic obial assimila ion o [
13
C]-labeled subs a es esul s in he
inco po a ion o hea y ca bon iso opes in o hei biomass, e.g., nucleic acids. As a
consequence, he buoyan densi y o DNA and RNA inc eases (Meselson and S ahl
1958). To al nucleic acids a e ex ac ed and RNA is sepa a ed om DNA. [
13
C]-
labeled ‘hea y’ RNA is hen sepa a ed om he ‘ligh ’ [
12
C]-RNA by isopycnic
ul acen i uga ion and subsequen ac iona ion o he g adien s. RNA o e e y
g adien ac ion is p ecipi a ed and e e sely ansc ibed in o complemen a y DNA
(cDNA). CDNA is used o iden i ica ion o RFs ela ed o [
13
C]-labeled o ganisms by
molecula me hods (e.g., RFLP inge p in ing [2.5.11], cDNA based gene lib a ies
[2.5.12]). Howe e , i is likely ha ‘hea y’ and ‘ligh ’ RNA a e no ully sepa a ed
du ing cen i uga ion. This may be due o insu icien labeling o ‘hea y’ RNA o co-
mig a ion o unlabeled ‘ligh ’ RNA wi h high G+C con en s (causing highe buoyan
densi ies) owa ds he ‘hea y’ ac ions (Lüde s e al. 2004; Mane ield e al. 2002a;
Rangel-Cas o e al. 2005). Combina ion o di e en molecula ools (e.g., RFLP and
cloning) should be aken in o accoun o a oid alse in e p e a ion o esul s only
gained by one me hodological app oach (Ben and Fo ney 2008).
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Figu e 6. Schema ic p o ocol o s able iso ope p obing (SIP). See ex o de ails.
2.5.6.1. Densi y G adien Cen i uga ion
A g adien solu ion (buoyan densi y: 1.790 ± 0.005 g ml
-1
) was p epa ed ha
con ained 4.61 ml caesium i luo oace a e (CsTFA, buoyan densi y:
2.0 ± 0.05 g ml
-1
, GE Heal hca e, Buckinghamshi e, UK), 0.175 ml o mamide and a
a iable amoun o g adien bu e (100mM KCl, 100 mM T is, 1 mM EDTA, pH 8).
0.8 – 1.2 ml o g adien bu e we e added un il he g adien solu ion had a buoyan
densi y o 1.790 ± 0.005 g ml
-1
. Densi y o he g adien solu ion was de e mined a
25°C. 500 ng o RNA we e mixed wi h 1 ml o g adien solu ion and ans e ed in o a
4.9-ml-cen i uga ion ube (Op iSeal Polyallome Tube, 13 x 48 mm; Beckmann, Palo
Al o, USA). The ubes we e illed wi h g adien solu ion, balanced un il a maximal
di e ence o 0.05 g was achie ed and closed wi h a plug. Tubes we e placed in an
ul a- e ical VTi 65.2 Ro o Package supplied wi h aluminium space s (Beckham
Coul e , Fulle on, USA). Sc ews we e closed wi h a o que w ench adap e (200
inch-pounds, Beckham Coul e , Fulle on, USA) o a bol ing o que o 60 inch-
pounds. [
12
C]- and [
13
C]-RNA we e sepa a ed by isopycnic cen i uga ion a 130,000
x g (37,800 pm) a 20°C o 67 hou s in a LE-70 ul a cen i uge (Beckman Coul e ,
Fulle on, USA). The o o an ou non-b aked. All g adien s we e se up wi h he
same g adien solu ion o minimize po en ial di e ences ha migh o he wise occu
by he use o g adien solu ions wi h simila bu non iden ical densi ies. Isopycnic
g adien s p epa ed om soil ea men s (2.3.1) con aining he same saccha ide
(including bo h he oxic and anoxic ea men s) we e cen i uged simul aneously. In
o al, 45 g adien s (15 o cellulose-, 15 o cellobiose-, and 15 o glucose-
supplemen ed ea men s) we e p ocessed in h ee cen i uga ion uns. A ube
con aining g adien solu ion wi hou RNA (= Blank) was en ained in e e y un o
measu e he densi y o he ac ions o he g adien s (2.5.6.3).
Rela i e Fluo escence
88 bp
90 bp
105 bp
F agmen Leng h
126 bp
Ex ac ion o
o al RNA
[
13
C]-RNA
[
12
C]-RNA
Incuba ion wi h
[
13
C]-Subs a e
Densi y
G adien
Cen i uga ion
RFLP Analyses
Re e se
T ansc ip ion o
RNA in o cDNA
Iden i ica ion o
[
13
C]-assimila ing
Mic oo ganisms
Collec ion o [
13
C]-
RNA and [
12
C]-RNA
by F ac iona ion
Inco po a ion o
[
13
C] in Nucleic
Acids o Ac i e
Mic oo ganisms
Molecula Analysis
o [
13
C]-RNA and
[
12
C]-RNA
16S RNA cDNA Gene Lib a ies
Rela i e Fluo escence
88 bp
90 bp
105 bp
F agmen Leng h
126 bp
Rela i e Fluo escence
88 bp
90 bp
105 bp
F agmen Leng h
126 bp
Ex ac ion o
o al RNA
Ex ac ion o
o al RNA
[
13
C]-RNA
[
12
C]-RNA
[
13
C]-RNA
[
12
C]-RNA
[
13
C]-RNA
[
12
C]-RNA
Incuba ion wi h
[
13
C]-Subs a e
Densi y
G adien
Cen i uga ion
RFLP Analyses
Re e se
T ansc ip ion o
RNA in o cDNA
Iden i ica ion o
[
13
C]-assimila ing
Mic oo ganisms
Collec ion o [
13
C]-
RNA and [
12
C]-RNA
by F ac iona ion
Inco po a ion o
[
13
C] in Nucleic
Acids o Ac i e
Mic oo ganisms
Molecula Analysis
o [
13
C]-RNA and
[
12
C]-RNA
16S RNA cDNA Gene Lib a ies
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2.5.6.2. F ac iona ion o G adien s
F ac iona ion o he g adien s was pe o med manually by a suc ion pump
(Econo Pump1, BioRad, He cules, USA) (Mane ield e al. 2002a). The cen i uga ion
ubes we e e ically ixed in a ag. A silicon hose (inne diame e : 1.6 mm) ha was
linked o he suc ion pump was equipped wi h a s e ile needle (23G x 1’’) and
inse ed ca e ully in o he ube unde nea h he plug. A second needle was used o
d ill a hole in o he bo om o he ubes. Ou lowing g adien solu ion was collec ed
a e he suc ion pump was s a ed ( low a e: 0.45 ml min
-1
) and s e ile wa e
(colou ed wi h b illian blue) was pumped on o he g adien solu ion (Figu e 7).
Ele en ac ions (~400 µl each) we e collec ed in 60-second-in e als om e e y
g adien . The ele en h ac ion was con amina ed wi h colou ed wa e and he e o e
disca ded om he analyses.
Figu e 7. F ac iona ion o g adien s.
See ex o de ails.
2.5.6.3. Measu emen o he Densi y o he F ac ions
The buoyan densi y o ac ions 1 – 10 was de e mined in he blank g adien
(i.e., wi hou RNA) o each cen i uga ion un (Mane ield e al. 2002b). The ac ions
we e empe ed o 25°C in a wa e ba h and ex ensi ely mixed. The weigh o he
ubes was measu ed on an analy ical balance (Analy ic AC 120 S, Sa o ius,
Ga ching, Ge many) be o e and a e 100 µl o he ac ion we e emo ed. The
densi y o e e y ac ion was de e mined 10 imes. Two ac ions ha p esumably
ep esen ed labeled ‘hea y’ RNA (co esponding o ac ion numbe s h ee and ou ,
buoyan densi y 1.813 ± 0.001 – 1.821 ± 0.005 g ml
-1
) we e pooled. Fu he mo e, wo
ac ions ha ep esen ed unlabeled ‘ligh ’ RNA (co esponding o ac ion numbe s
eigh and nine, buoyan densi y 1.767 ± 0.000 – 1.776 ± 0.000 g ml
-1
) (Mane ield e
al. 2002b) we e pooled. Pooled ac ions we e used o u he analyses.
M
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2.5.6.4. RNA P ecipi a ion
RNA in ‘ligh ’ and ‘hea y’ ac ions was p ecipi a ed acco ding o Degelmann e
al. 2009a. 200 µl o each ac ion we e mixed wi h 130 µl NaCl (3 M, pH 5.2), 13.6 µl
glycogen (10 mg ml
-1
), and 1,000 µl ice cold e hanol (96%). Samples we e incuba ed
o a leas 12 hou s a -20°C. RNA was p ecipi a ed by cen i uga ion (13,000 x g,
20 minu es, 4°C). The supe na an was disca ded, he RNA pelle washed wi h 500 µl
ice cold e hanol (70% in RNAse- ee ddH
2
O, -20°C), and d ied a oom empe a u e.
The RNA was dissol ed in 22 µl RNAse- ee ddH
2
O and 2 µl o RNA solu ion we e
used o quan i ica ion by luo escence based me hods (2.5.4.2). RNA samples we e
di ec ly used o e e se ansc ip ion o RNA in o cDNA (2.5.7).
2.5.7. Re e se T ansc ip ion o RNA in o cDNA
Re e se ansc ip ase o RNA in o single-s anded complemen a y DNA (cDNA)
was pe o med wi h he Supe Sc ip VILO cDNA Syn hesis Ki o he Supe Sc ip III
Fi s -Syn hesis Sys em (bo h: In i ogen, Da ms ad , Ge many). Random hexame s
(50 ng µl
-1
) we e used in all eac ions. Re e se ansc ip ion PCR (RT-PCR) was
conduc ed on a TG adien The mocycle (Biome a, Gö ingen, Ge many). Samples
we e p epa ed acco ding o he manu ac u e ’s p o ocols. Annealing o andom
hexame s o RNA was pe o med o 10 minu es a 25°C, ollowed by 90 minu es a
42 – 50°C o cDNA syn hesis. Reac ions we e e mina ed by hea -inac i a ion o he
enzyme o 5 minu es a 85°C. A nega i e con ol (RNase- ee ddH
2
O) and a posi i e
con ol (con ol RNA supplied by he manu ac u e ) we e en ained in e e y e e ese
ansc ip ion. CDNA was s o ed a -80°C.
2.5.8. In Vi o T ansc ip ion o DNA in o RNA
Single-s anded RNA was p epa ed by in i o ansc ip ion o pGEM-T ec o
DNA wi h he T7 ansc ip ion ki (Fe men as, S .Leon-Ro , Ge many). Single-
s anded RNA was used o co ec ion o ansc ip numbe s measu ed in qPCR
assays (2.5.10). Vec o DNA was ampli ied by M13-PCR (2.5.9.3). The PCR p oduc
was pu i ied wi h Millipo e PCR
96
Cleanup Pla es (Millipo e Coope a ion, Bed o d,
USA) (2.5.3.3) and quan i ied wi h he Quan -iT-PicoG een dsDNA eagen Ki
(2.5.4.2). A maximum o 1 µg o DNA was used o in i o ansc ip ion acco ding o
he manu ac u e ’s desc ip ion. A con ol DNA included in he ki was en ained in he
analysis. In i o ansc ip ion was pe o med by 2 hou s o incuba ion a 37°C and
eac ion was s opped by eezing samples a -20°C. Success ul ansc ip ion was
checked by aga ose gel elec opho esis (2.5.5). Templa e DNA was emo ed by
DNAse I diges ion (2.5.2.2). Remaining RNA was quan i ied wi h he RiboG een
Quan i a ion Reagen Ki (2.5.4.2).
2.5.9. Polyme ase Chain Reac ion (PCR)
Polyme ase chain eac ion (PCR) is a me hod o mul iply agmen s o DNA o
cDNA by se e al o de s o magni ude, i.e., i gene a es mul iple copies o a pa icula
gene sequence. The p ocess o enzyma ic ampli ica ion o hese gene agmen s can
be di ided in o h ee s eps: dena u a ion o he DNA empla e, annealing o gene-
speci ic p ime s o single s anded DNA, and elonga ion o he complemen a y s and
by a he mally s able DNA-polyme ase (Saiki e al. 1985; Saiki e al. 1988).
M
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Name o
P ime Posi ion
a
Sequence
(5´-3´)
b
T
m
(°C)
c
Ta ge genes
o Re e ence
Sph1R 670 – 685 CTG TCA ATT
CCG CCT 42 ‘Sphingo1-4’
e
his wo k
Deh1R 580 – 600 CGA CTT GAA
CGA CCG CCT 53 ‘Deha1’
e
his wo k
DNA Inhibi ion Co ec ion Assay
T7P omF n.a.
TAA
TAC
GAC
TAT AGG G 38 pGEM-T ec o
inse Degelmann
e al. 2009b
M13Re R n.a.
CAG
GAA
ACA
GCT ATG ACC 48 pGEM-T ec o
inse Degelmann
e al. 2009b
RNA Inhibi ion Co ec ion Assay
IhcF n.a.
ATT
GGG
CCC
GAC GTC 47 pGEM-T ec o
inse Wieczo ek e
al. 2011
IhcR n.a.
ATT
TAG
GTG
ACA CTA TAG
AAT A
46 pGEM-T ec o
inse Wieczo ek e
al. 2011
a
Numbe s in he names o p ime s indica e he 5’-posi ion o binding si es ela i e o he
e e ence sequence o Esche ichia coli [ACCX80725]. Abb e ia ions: F, o wa d p ime ; R,
e e se p ime .
b
Y, C o T (IUPAC, In e na ional Union o Pu e and Applied Chemis y).
c
T
m
(basic mel ing empe a u e) o p ime s was calcula ed online wi h OligoCalc:
h p://www.basic.no hwes e n.edu/bio ools/oligocalc.h m]; las isi : 15.07.2010; Kibbe 2007
d
Bold and unde lined bases in p ime sequence o amily-le el axa indica e misma ches o
non- a ge con ol (3.4).
e
Names and a ilia ion o amily-le el axa acco ding o (3.2.4.2).
n.a., Non-applicable.
P ime s used in qPCR eac ions (Table 15) we e pu chased om Biome s
GmbH (Biome s.ne , Ulm, Ge many). PCR eac ions we e p epa ed in 20 µl olumes
wi h PCR chemicals o Bioline (Luckenwalde, Ge many) (Table 16) and pe o med on
an iQ5 iCycle (Bio-Rad, He cules, USA).
M
ATERIAL
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Table 16. Chemical composi ion o qPCR eac ions.
Reagen s
(Conc.)
Domain-le el
assays Family-le el
assays
Inhibi ion
co ec ion
assays
Volume
[µl]
Final
conc. Volume
[µl]
Final
conc. Volume
[µl]
Final
conc.
SensiMix SYBR
& Fluo escein
(2x)
a
10 1 x 10 1 x 10 1 x
Fo wa d
p ime
b, c
(10 µM)
1.5 0.75 µM
1 – 1.5
0.5 –
0.75 µM
1 0.5 µM
Re e se
p ime
b, c
(10 µM)
1.5 0.75 µM
1.5 – 3
0.75 –
1.5 µM 1 0.5 µM
Templa e DNA
o cDNA 5.0 - 5.0 - 5.0 -
PCR-H
2
O Ad 20 - Ad 20 - Ad 20 -
a
SensiMix SYBR & Fluo escein (2 x; Bioline, Luckenwalde, Ge many) con ains eac ion
bu e , hea -ac i a ed DNA Polyme ase, ul a-pu e dNTPs, 6 mM MgCl
2
, s abilize s,
Fluo escein, and SYBR® G een I.
b
De ails abou p ime s in Table 15.
c
The inal concen a ion o each p ime in amily-le el assays numbe 1, 3, 5 and 8 was
750 nM (Table 25; 3.4). The inal concen a ion o each p ime in amily-le el assays numbe
2, 4, 6 and 7 was 500 nM ( o wa d p ime ) and 1500 nM ( e e se p ime ) (Table 25; 3.4).
conc., Concen a ion
The mal p o ocols a e summa ized in Table 17. De ails abou amily-le el
assays a e gi en in he esul s sec ion (3.4). The luo escence signal was eco ded
du ing elonga ion. Subsequen aga ose gel elec opho esis (2.5.5) was used o
check o he o ma ion o unspeci ic bands. Speci ici y o domain- and amily-le el
assays was analyzed by sequencing o qPCR amplicons (2.5.10.2).
M
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Table 17. The mal p o ocols o ampli ica ion o 16S RNA genes and clone
inse sequences.
P ime s pai s
a
Family-
le el
assays
b
Gm5F /
Eub534R A c344F /
A ch915R IhcF /
IhcR T7P omF /
M13Re
No.
S ep T (°C) / ime (minu es:seconds)
1 Ini ial
dena u a ion 94 / 8:00 94 / 8:00 94 / 8:00 94 / 8:00 94 / 8:00
S ep 2 – 4 35 x 35 x 45 x 38 x 38 x
2 Dena u a ion
95 / 0:30 95 / 0:30 95 / 0:30 95 / 0:30- 95 / 0:30-
3 Annealing 56 – 67.5
b
/ 0:40 55.7 / 0:40 55.7 / 0:40 60 / 0:15 61.2 / 0:15
4 Elonga ion 72 / 0:40 72 / 0:30 72 / 0:30 72 / 0:45 72 / 0:45
5 Final
Elonga ion 72 / 5:00 72 / 5:00 72 / 5:00 72 / 7:00 72 / 10:00
161 x 49 x
6 Mel ing
cu e 70 – 94 / 0:10
(+0.15°C / cycle)
70 – 94 / 0:10
(+0.5°C / cycle)
Leng h o
amplicon (bp) See Table
25 190 570 450 450
a
De ails abou p ime s in Table 15.
b
De ails abou p ime pai s, app op ia e annealing empe a u e, and leng h o amplicon o
amily-le el assay in he esul s sec ion (3.4).
bp, Base pai s.
No., Numbe .
2.5.10.2. P epa a ion o Quan i a i e DNA S anda ds
Each qPCR was calib a ed wi h quan i a i e assay-speci ic DNA s anda ds.
These s anda ds we e p epa ed wi h amily-le el 16S RNA gene sequences e-
ampli ied (p ime pai : 27F/907RM, Table 11) om he co esponding clone inse
sequence (2.5.9.3, 3.4). PCR p oduc s we e pu i ied wi h Millipo e PCR
96
Cleanup
Pla es (Millipo e Coope a ion, Bed o d, USA) (2.5.3.3) and quan i ied wi h he Quan -
iT-PicoG een dsDNA eagen Ki (In i ogen, Ka ls uhe, Ge many) (2.5.4.2). The
numbe o a ge sequences c
TS
was calcula ed wi h Equa ion 7, including he
concen a ion o pu i ied s anda d DNA, he leng h (i.e., he numbe o bases) o he
amplicon, he molecula weigh o one base pai in double s anded DNA, and he
A ogad o cons an N
A
.
M
ATERIAL
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Equa ion 7. Numbe o a ge molecules in pu i ied s anda d solu ions.
A
bpbases
DNAs d
TS
N
MWN
c
c•
•
=
c
TS
, numbe o a ge sequences ( a ge s µl
-1
); c
DNAS d
, concen a ion o s anda d DNA (ng
DNA
µl
-1
); N
bases
, leng h o he amplicon; MW
bp
, molecula weigh o one base pai in double-
s anded DNA (MW
bp
= 650 ng nmol
-1
); N
A
, A ogad o cons an (N
A
= 6.23 × 10
23
molecules
nmol
-1
).
DNA s anda ds we e dilu ed wi h DEPC-H
2
O o 2 × 10
9
a ge molecules µl
-1
and s o ed a -80°C. Di ec ly be o e use, dilu ion se ies we e p epa ed o achie e
concen a ions o 10
7
– 10
0
molecules pe qPCR eac ion by applica ion o 5 µl o
dilu ed s anda d DNA as empla e (2.5.10.1). DNA s anda ds o quan i ica ion o
Bac e ia and A chaea we e supplied by Ka ha ina Palme , Depa men o Ecological
Mic obiology, Uni e si y o Bay eu h.
2.5.10.3. E alua ion o he Speci ici y o Assays in Soil Samples
Se e al ac o s we e aken in o accoun o e alua e a ge g oup speci ici y o
amily-le el and domain-le el qPCR assays. Mel cu es o PCR p oduc s o qPCR
s anda ds we e compa ed wi h PCR p oduc s ampli ied om soil samples.
Subsequen ly, he o ma ion o unspeci ic bands was checked by aga ose gel
elec opho eses (2.5.5). QPCR p oduc s om each assay o e e y sample (i.e., all
eplica es, ime poin s, and subs a es) o ea men s in sel -cons uc ed incuba ion
chambe s (2.3.2) we e pooled and sen o LGC Genomics (Be lin, Ge many) o
cloning and sequencing (2.5.13). 17 – 24 sequences pe assay we e analysed wi h
BLAST (Al schul e al. 1990; 2.5.14).
2.5.10.4. Co ec ion o PCR Inhibi ion and Calcula ion o T ansc ip Numbe s
Co-ex ac ed compounds (e.g. humic acids) o a iable e e se ansc ip ion
e iciencies may impac on qPCR measu emen s. This migh lead o unde es ima ion
o ansc ip numbe s (Degelmann e al. 2009b; Wieczo ek e al. 2011). Thus,
ansc ip numbe s we e co ec ed by measu ing inhibi ion ac o s in all samples
(Degelmann e al. 2009b; Wieczo ek e al. 2011). A pheA (gene ha encodes a
phenol hyd oxylase) con aining pGEM-T ec o inse was used as inhibi ion con ol
DNA and as empla e o p epa a ion o con ol RNA (2.5.8). E e y en i onmen al
DNA and RNA sample was spiked wi h 2 × 10
9
inhibi ion con ol molecules µl
-1
.
Spiked RNA ex ac s we e ansc ibed in o cDNA (2.5.7), and all ex ac s we e
quan i ied wi h domain-, amily-le el, and DNA o RNA inhibi ion assays (Table 15
and Table 17). Co ec ed 16S RNA gene ansc ip numbe s (lg[SQ
co ec ed
]) o
Bac e ia, A chaea and amily-le el axa we e calcula ed wi h Equa ion 8.
M
ATERIAL
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50
Equa ion 8. Co ec ion o ansc ip numbe s by inhibi ion co ec ion assays.
••=
INHIBCORR
Assay
isINHIBCORR
se INHIBCORR
measu edco ec ed
E
E
SQ
SQ
SQSQ )lg(
)lg(
lg)lg(
_
_
lg(SQ
co ec ed
), co ec ed loga i hmic alues o he s a ing quan i y (SQ) measu ed in domain-
o amily-le el assays; SQ
measu ed
, s a ing quan i y measu ed in domain- o amily-le el
assays; lg(SQ
INHIBCORR_se
), loga i hmic alues o he s a ing quan i y o con ol DNA o RNA
spiked in o en i onmen al ex ac s; lg(SQ
INHIBCORR_is
), loga i hmic alues o he s a ing
quan i y o con ol DNA o RNA measu ed; E
Assay
, ampli ica ion e iciency (E) o domain- o
amily-le el assay; E
INHIBCORR
, ampli ica ion e iciency o he Inhibi ion co ec ion assays.
Family-le el assays (3.4) did no yield su icien gene ampli ica ion in DNA
samples (i.e., a ge sequences we e below he de ec ion limi o abou 10 genes pe
eac ion; C
T
> 30). P esen ed ansc ip numbe s in his s udy e e exclusi ely o
ansc ip numbe s quan i ied in cDNA samples. The o al amoun o RNA ha was
ex ac ed om expe imen al eplica es was simila , bu a iable (Figu e 9). Thus,
co ec ed 16S RNA ansc ip numbe s we e quan i ied pe ng RNA a he han pe
g am d y weigh o soil. These absolu e numbe s o ansc ip s o amily-le el axa
we e di ided by he co esponding numbe o ansc ip s o o al Bac e ia o u he
co ec o a iabili y in lysis e iciencies and he g ow h o Bac e ia. Fo each
eplica e, hese copy numbe s o amily-le el axa we e no malized based on he
highes copy numbe measu ed (i.e., he highes numbe ob ained o each assay,
including subs a e-supplemen ed and unsupplemen ed mic ocosms, was se a
100%) and a e gi en in pe cen . Da a p esen ed a e means o expe imen al
iplica es (including s anda d de ia ions) o duplica es (wi hou s anda d de ia ion).
Figu e 9. To al RNA ex ac ed om each sampling poin o each ea men .
Filled ba s, CMC-supplemen ed ea men s; Shaded ba s, cellobiose-supplemen ed
ea men s; Emp y ba s, unsupplemen ed con ols. E o ba s indica e s anda d de ia ions
(n = 3).
Time [h]
0 48 102 168 192
ng RNA g
DW-1
0
500
1000
1500
2000
2500
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2.5.11. Te minal Res ic ion F agmen Leng h Polymo phism ( RFLP)
Analysis
TRFLP is a PCR-based inge p in ing me hod ha allows o he apid
cha ac e iza ion o mic obial communi ies in a ious en i onmen s (Liu e al. 1997;
Thies 2007). 16S RNA o unc ional genes a e ampli ied wi h luo escence labeled
p ime s (one o bo h), esul ing in e minal-labeled PCR p oduc s (Figu e 10).
Diges ion o hese PCR p oduc s wi h a es ic ion endonuclease ( ype II) yields
e minal-labeled es ic ion agmen s ( RFs). These agmen s a e subsequen ly
leng h sepa a ed on a polyac ylamide gel (PAGE) (Liu e al. 1997). Di e en
mic obial communi ies esul in di e en RF p o iles in which e e y RF is indica i e
o a leas one communi y membe (Ma sh 2005). In silico diges ion o ibosomal
da abase sequences o clone inse sequences ob ained om he same
en i onmen al sample allows he a ilia ion o single RFs o mic obial axa.
Figu e 10. Scheme o RFLP analysis.
See ex o de ails.
In his s udy, 16S RNA cDNA gene based RFLP was pe o med o apidly
compa e bac e ial and a chaeal soil communi ies in ol ed in he consump ion o
supplemen ed ca bon (i.e., cellulose, cellobiose, glucose; 2.5.6). TRF p o iles we e
u he used o iden i y sequences o [
13
C]-labeled o ganisms in gene lib a ies.
Al hough cDNA o ‘hea y’ ac ions was used o cloning, unlabeled geno ypes (i.e.,
16S RNA cDNA sequences) in ‘hea y’ ac ions had high G+C con en s (53 – 61%,
da a no shown) and may ha e hus been un olded RNA molecules yielding buoyan
densi ies simila o labeled geno ypes (Lüde s e al. 2004). The e o e, RF p o iles o
Bac e ia and A chaea we e gene a ed om cDNA samples de i ed om he ‘ligh ’
and ‘hea y’ ac ion o [
12
C]- and [
13
C]-s able iso ope p obing g adien s (2.5.6.3,
2.5.7), and compa ed. This minimizes o e es ima ion o he ac i e pa o he
cDNA o
‘Hea y‘ o
‘Ligh ‘ F ac ion
16S RNA Gene Ampli ica ion
wi h Fluo escence Labeled
P ime s
Diges ion wi h
Res ic ion Enzyme PAGE Compa ison o
RF P o iles
Rela i e Fluo escence
74 bp
90 bp
F agmen Leng h
[
13
C]
Rela i e Fluo escence
108 bp
F agmen Leng h
123 bp
[
12
C]
MspI, TaqI
MspI, TaqI
Te minal Labeled
Amplicons Sepa a ion
o RFs In silico Iden i ica ion
o Labeled RFs
Sequence-
dependen RFs
cDNA o
‘Hea y‘ o
‘Ligh ‘ F ac ion
16S RNA Gene Ampli ica ion
wi h Fluo escence Labeled
P ime s
Diges ion wi h
Res ic ion Enzyme PAGE Compa ison o
RF P o iles
Rela i e Fluo escence
74 bp
90 bp
F agmen Leng h
Rela i e Fluo escence
74 bp
90 bp
F agmen Leng h
[
13
C]
Rela i e Fluo escence
108 bp
F agmen Leng h
123 bp
Rela i e Fluo escence
108 bp
F agmen Leng h
123 bp
[
12
C]
MspI, TaqI
MspI, TaqI
Te minal Labeled
Amplicons Sepa a ion
o RFs In silico Iden i ica ion
o Labeled RFs
Sequence-
dependen RFs
M
ATERIAL
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ETHODS
52
mic obial communi y. TRF p o iles o day 0 (co esponding o he au och honous soil
communi y) we e gene a ed exclusi ely o [
13
C]- ea men s, since he same
homogenized soil samples was used in [
12
C]- ea men s. P ime pai s, PCR
condi ions, and he mal p o ocols as men ioned abo e (Table 11 - Table 13).
2.5.11.1. Mung Bean Endonuclease Diges ion
P ema u ed e mina ion o he elonga ion s ep du ing PCR can lead o single-
s anded pa s o he amplicons. This, in consequence, migh p omo e he o ma ion
o ‘pseudo- RFs’, i.e., RFs ha esul om alse es ic ion diges ion a non- e minal
es ic ion si es (Ege and F ied ich 2003). Single-s anded ex ensions a he
e minal ends we e emo ed wi h mung bean endonuclease diges ion (New England
Biolabs, Ipswich, USA). 50 µl o pu i ied PCR p oduc we e mixed wi h 5.5 µl eac ion
bu e NEB2 (10x) and 2 µl mung bean endonuclease (0.5 U µl
-1
). Incuba ion was
pe o med a 30°C o 1 hou . Endonuclease diges ion was s opped by pu i ica ion o
samples wi h Millipo e PCR
96
Cleanup Pla es (Millipo e Coope a ion, Bed o d, USA)
(2.5.3.3).
2.5.11.2. Res ic ion Diges ion
Subsequen es ic ion diges ion was pe o med wi h MspI (5’ 3’ es ic ion
si e: C^CGG; New England Biolabs, Ipswich, USA, bac e ial 16S RNA genes) o
TaqI (5’ 3’ es ic ion si e: T^CGA; New England Biolabs, USA; a chaeal 16S RNA
genes) (Degelmann e al. 2009b). 7 µl pu i ied PCR p oduc we e mixed wi h 1 µl
eac ion bu e NEB2 (10x; MspI) o 1 µl NEB3 (10x, TaqI), 1 µl BSA (10x), and 1 µl
es ic ion enzyme (2 U µl
-1
). Res ic ion diges ion was pe o med a 37°C o 4 hou s
(MspI) o a 65°C o 3 hou s ( TaqI). Reac ions we e s opped by hea inac i a ion o
he enzyme o 5 minu es a 95°C (MspI) o 20 minu es a 80°C (TaqI). Remaining
DNA was quan i ied wi h he Quan -iT-PicoG een dsDNA eagen Ki (In i ogen,
Ka ls uhe, Ge many) (2.5.4.2) and dilu ed wi h PCR-H
2
O o a concen a ion o
0.5 ng µl
-1
.
2.5.11.3. Dena u ing Polyac ylamide Gelelec opho esis (PAGE)
Dena u ing gel elec opho esis was pe o med on a NEN model 4300 DNA
analyze (Lico , Lincoln, USA) as desc ibed (Hambe ge e al. 2008). Gel pla es
(Bo o loa glass pla es 25 mm x 25 mm x 5 mm, LICOR, Lincoln, USA) we e
ho oughly cleaned sequen ially wi h ddH
2
O, e hanol (70%) and isop opanol (80%).
Fo s abiliza ion o he gel pocke s 250 µl bind-silane solu ion (1:1 bind silane
[plusOne; GE Heal hca e, Pisca away, USA] and 10% CH
3
COOH) was applied o he
pla es a he a ea a which he comb has o be inse ed. The pla es we e sepa a ed
by space s (0.2 mm ick), ixed wi h assembly ails and cas ing pla e, and placed in
he gel cas ing s and. The polyac ylamide gel was p epa ed by gen ly mixing 15 g
u ea (Roche Pha ma, Reinach, Swi ze land), 3.75 ml o 40% ac ylamide-bis solu ion
(37.5 : 1; 2.6% C; Bio-RAD, He cules, USA), 6 ml 5 x TBE bu e (450 mM T is,
450 mM H
3
BO
3
, 10 mM EDTA, pH 8 [Samb ook and Russell 2001]), and 9.25 ml
ddH
2
O. The gel solu ion was il e ed (∅ 0.2 µm) o emo e excess undissol ed sal s.
The polyme isa ion eac ion was s a ed by addi ion o 175 µl ammonium pe sul a e
(APS, 440 mM) and 17 µl N,N,N,N-Te ame hyle hylendiame (TEMED, ul a-pu e;
In i ogen, Ka ls uhe, Ge many). The gel was pou ed and a ec angula oo h comb
(48 lanes) was inse ed. Polyme isa ion was ca ied ou a 25°C o 45 minu es. The
M
ATERIAL
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M
ETHODS
53
glass pla es we e placed in o he DNA analyse , he bu e anks we e ixed, and
illed wi h 1 x TBE bu e . The comb was emo ed and gel pocke s we e cleaned om
p ecipi a ed u ea by gen ly lushing wi h bu e . Be o e samples we e loaded on o he
gel a p e- un was pe o med a 1,200 V and 45°C o 25 minu es. Samples we e
p epa ed o elec opho esis by mixing 2 µl o sample wi h 2 µl S op-Solu ion (Lico ,
Lincoln, USA), dena u a ion a 95°C o 3 minu es, and placing on ice. A 50 – 700 bp
leng h s anda d (Size S anda d IRDye 700; Lico Biosciences o mic oSTEP-24a
(700); Mic ozone L d, Haywa ds Hea h, UK) was p ocessed analogous. 0.5 µl size
s anda d o 0.8 µl sample we e loaded on o he gel and elec opho esis was
pe o med a 1,200 V and 45°C o 4 hou s.
2.5.11.4. Analysis o RFLP P o iles
Scanned gel images we e expo ed and analysed wi h GelQues (Ve sion
2.6.3.; Sequen ix, Klein Raden, Ge many). This p og am gene a es RFLP p o iles in
which e e y peak ep esen s one RF. The peak heigh o a RF is di ec ly linked o
he ela i e in ensi y o he luo escence label. To compa e ela i e numbe s, he
luo escence alue o a de ec ed RF was no malized agains he luo escence alue
o he espec i e RF in he RFLP p o ile wi h he lowes o al luo escence. TRFs
wi h alues below 5% we e excluded. Means and s anda d de ia ions we e
calcula ed o
end
measu emen s o [
13
C]-supplemen ed ea men s (2.3.1). The
RFLP p o iles de i ed om ‘hea y’ RNA ac ions o [
13
C]- and [
12
C]- ea men s we e
compa ed o iden i ying RFs o labeled geno ypes. The RFs we e sco ed as
labeled when hey we e p esen only in [
13
C]-p o iles o when he ela i e in ensi y in
[
13
C]-p o iles was highe han in [
12
C]-p o iles a he same espec i e ime in e al.
This p ocedu e a oided an o e es ima ion o labeled geno ypes, as unlabeled
geno ypes may co-mig a e owa ds he ‘hea y’ RNA ac ion (Lüde s e al. 2004;
Mane ield e al. 2002a; Rangel-Cas o e al. 2005). TRFs we e assigned o pu a i e
axa by in silico analyses o clone inse sequences (2.5.12) and ibosomal
da abases using he so wa e ool TRFCUT included in ARB (Ricke e al. 2005;
2.5.14.3).
2.5.12. Cons uc ion o Gene Lib a ies
Gene lib a ies a e cons uc ed by inse ion o o eign DNA (e.g. PCR p oduc s
o ce ain genes) in o a cloning ec o (liga ion) and u he in oduc ion o his ec o
( ans o ma ion) in o compe en hos cells (Samb ook and Russell 2001; Yansich-
Pe on e al. 1985). Cul i a ion o hos cells yields clones (i.e., colonies) o which he
ones a e picked ha appa en ly ca y cloning ec o s wi h inse ed DNA (blue/whi e
sc eening). 16S RNA cDNA genes o each Bac e ia, A chaea, and amily-le el axa
ampli ied by qPCR (2.5.10, 2.3.2) we e pooled (i.e., all eplica es, ime poin s, and
subs a es). Cloning o bac e ial 16S RNA genes ampli ied om cDNA o he ‘hea y’
ac ion o [
13
C]-g adien s (2.3.1, 2.5.9.2) was pe o med wi h he pGEM-T ec o
sys em II (P omega, Madison, USA) a e he manu ac u e ’s p o ocol.
2.5.12.1. Liga ion
The pGEM-T ec o (size: ~3000 bp) is linea ized wi h a single 3’- e minal
hymidine a bo h ends. This p e en s eci cula iza ion o he ec o and imp o es he
e iciency o liga ion o he PCR p oduc in o he ec o (due o a polyme ase-
media ed adenosine-o e hang a he 3’ end o he DNA s and; Mezei and S o s
M
ATERIAL
A
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M
ETHODS
54
1994; Robles and Doe s 1994). The ec o con ains a gene o ampicillin- esis ance
and a MCS (mul iple cloning si e) ha is loca ed in he lacZ ope on. lacZ encodes o
he enzyme β-galac osidase and i s ope on is dis up ed by inse ion o a o eign DNA
a he MCS. Thus, lacZ canno be exp essed in hos cells ha ook up a plasmid wi h
DNA inse . The inabili y o such clones o p oduce β-galac osidase can be used o
selec ion o inse posi i e clones (2.5.12.3). The op imal inse o ec o mola a io
o he pGEM-T ec o sys em is deno ed o be 1:1, bu success ul liga ion can also
be achie ed be ween a ios o 8:1 and 1:8. The app op ia e amoun o inse o
include in he liga ion eac ion can be calcula ed o di e en a ions (Equa ion 9). A
a io be ween 1:1 and 3:1 was used in his s udy, wha co esponds o 15 – 45 ng o
PCR p oduc pe eac ion (Table 18).
Equa ion 9. Inse : ec o a io.
a io mola ec o : inse
ec o o size kb
inse o size kb •
•
=
ec o
inse
ng
ng
Pu i ied 16S RNA cDNA gene PCR p oduc s (2.5.3.2) we e inse ed in o he
ec o plasmid (pGEM-T) by a T4 DNA ligase. The liga ion eac ions we e p epa ed
(Table 15), mixed, placed in o a wa e ba h (25°C), and incuba ed o e nigh a 4°C.
The op imal empe a u e o liga ion was pe ambula ed du ing he cool down o he
wa e ba h.
Table 18. Liga ion eac ion mix.
Reac ion componen
(concen a ion) Amoun (µl) Final concen a ion
Rapid Liga ion Bu e (2 x)
a
5 1x
pGEM-T ec o (50 ng µl
-
1
) 1 5 ng µl
-
1
T4 DNA Ligase (3 Weiss U µl
-
1
) 1 0.3 U µl
-
1
PCR p oduc (inse DNA) 0.5 - 2 15 – 45 ng
PCR-H
2
O Ad 10 -
a
2x Rapid Liga ion Bu e con ains 60 mM T is-HCl (pH 7.8), 20 mM MgCl
2
, 20 mM DTT, 2
mM ATP, and 10% polye hylene glycol (MW8000, ACS G ade).
2.5.12.2. T ans o ma ion
Vec o plasmids we e inse ed in o high-e iciency compe en Esche ichia coli
JM 109 cells (> 10
8
c u µg
DNA-1
). E. coli cells we e pu chased om P omega
(Madison, USA) o p epa ed by Ral Me el a he Depa men o Ecological
Mic obiology (Uni e si y o Bay eu h) acco ding o he ollowing p ocedu e.
M
ATERIAL
A
ND
M
ETHODS
55
Liquid E. coli cul u es (in glyce ol) we e ans e ed on o LB-pla es and
incuba ed a 37°C o 12 hou s. One colony was picked, ans e ed in o 5 ml LB-
medium (2.1) and incuba ed o e nigh . 1 ml o e nigh -cul u e was used as inoculum
o 50 ml LB-medium and cul u es we e incuba ed a 37°C o abou 5 hou s un il an
op ical densi y (OD
660
) o 0.5 was eached. The cells we e collec ed by cen i uga ion
(4,000 pm, 4°C, 5 minu es), esuspended in 15 ml s e ile, ice-cold ans o ma ion
bu e 1 (10 mM CaCl
2
, 50 mM MnCl
2
⋅ 4 H
2
O, 30 mM po assium ace a e [CH
3
CO
2
K],
100 mM RbCl, 15% glyce ol, pH 5.8), and incuba ed on ice o 90 minu es.
Subsequen ly, cells we e collec ed by cen i uga ion and esuspended in 2 ml s e ile,
ice cold ans o ma ion bu e 2 (75 mM CaCl
2
, 10 mM MOPS, 10 mM RbCl, 15%
glyce ol, pH 8.0). Cells we e s o ed in 220 µl po ions a -80°C. Di ec ly be o e use
cells we e hawed on ice.
Fo ans o ma ion 220 µl compe en cells we e gen ly mixed wi h 2.5 µl liga ion
eac ion and mix u es we e incuba ed on ice o 30 minu es. Cells we e placed in a
p ehea ed wa e ba h (42°C) o 45-50 seconds (hea -shock) o o ce up aking o he
ec o plasmid. The cells we e immedia ely placed on ice o 2 minu es. 950 µl SOC-
medium (2.1) we e added and cells we e incuba ed a 37°C o 90 minu es on a
he momixe (Eppendo , Hambu g, Ge many) unde cons an slow shaking (300
pm). Subsequen ly, abou 400 µl o ans o med cells we e sp ead o e
LB/Ampicillin/IPTG/X-Gal-pla es (2.1) and incuba ed o e nigh a 37°C. The addi ion
o ampicillin o aga pla es exclusi ely allows g ow h o cells ha acqui ed he
ampicillin- esis ance gene by up ake o he ec o plasmid.
2.5.12.3. Blue/whi e Sc eening o Clones
Blue/whi e sc eening (Samb ook and Russell 2001) was used o selec o
clones ha do no only ca y a ec o plasmid (as selec ed by ampicillin esis ance),
bu also a plasmid wi h a DNA inse a he MCS. IPTG induces he exp ession o he
lac ope on and hence he p oduc ion o he enzyme β-galac osidase. This enzyme
eac s wi h he colou less compound X-Gal (lac ose analogue) and leads o he
o ma ion o he blue insoluble pigmen 5,5’-dib omo-4,4’dichlo o-indigo, causing a
cha ac e is ic blue colou in he colonies. Whi e colonies a e su icien o he
p oduc ion o β-galac osidase, indica ing ha he lacZ ope on is dis up ed by a DNA
inse . Whi e colonies we e andomly picked wi h a s e ile oo hpick, ans e ed on o
esh LB/Ampicillin/IPTG/X-Gal-Pla es (2.1), and incuba ed a 37°C o e nigh .
Selec ion o posi i e clones ha ca y ec o plasmids wi h pu a i e 16S RNA cDNA
gene inse sequences was pe o med by ampli ica ion o he inse DNA by M13-
PCR (2.5.9.3).
2.5.13. Sequencing
Pu i ied M13-PCR p oduc s o 16S RNA cDNA gene inse sequences (2.5.9.3)
we e sen o Mac ogen (Kumchun-ku Seoul, Ko ea) o sequencing. QPCR p oduc s
o domain- and amily-le el assays (2.5.10.3) we e sen o LGC Genomics (Be lin,
Ge many) o cloning and sequencing. M13uniF (Table 11) was used as sequencing
p ime .
R
ESULTS
62
Figu e 11. Deg ada ion o cellulose (A), cellobiose (B), and glucose (C) in soil slu ies.
Values a e he means o iplica es ([
13
C]-cellobiose, [
13
C]-
glucose), duplica es ([
13
C]-cellulose, [
12
C]-cellobiose, [
12
C]-glucose), o a single ea men ([
12
C]-cellulose). E o ba s indica e s anda d de ia ions.
Concen a ions o compounds in unsupplemen ed ea men s we e sub ac ed om concen a ions o p oduc s in supplemen ed ea men s; he di e ence is
shown. Solid symbols, alues om ea men s supplemen ed wi h [
13
C]-subs a es; Open symbols, alues om ea men s supplemen ed wi h [
12
C]-subs a es.
Symbols:
✖
cellobiose; glucose; ▼ ace a e; bu y a e;
✚
p opiona e; ● ca bon dioxide; ■ molecula hyd ogen; ▲ e ous i on. A ows indica e sampling o
s able iso ope p obing analyses.
0 10 20
0 10 20
0 10 20
0 10 20
0 4 8 12
0
10
20
30
40
50
0 4 8 12
0
10
20
30
40
50
0 40 80
0
10
20
30
40
50
0 40 80
0 40 80
0.0
0.2
0.4
0.6
0.0
0.2
0.4
0.6
0
2
4
6
8
10
0
2
4
6
8
10
0
2
4
6
8
10
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
PulsedSubs a e [mM]
B Cellobiose
C Glucose
Pulsed Subs a e [mM]
Time [d]
A Cellulose
OXIC ANOXIC
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
0 10 20
0 10 20
0 10 20
0 10 20
0 4 8 12
0
10
20
30
40
50
0 4 8 12
0
10
20
30
40
50
0 40 80
0
10
20
30
40
50
0 40 80
0 40 80
0.0
0.2
0.4
0.6
0.0
0.2
0.4
0.6
0
2
4
6
8
10
0
2
4
6
8
10
0
2
4
6
8
10
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
PulsedSubs a e [mM]
B Cellobiose
C Glucose
Pulsed Subs a e [mM]
Time [d]
A Cellulose
OXIC ANOXIC
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
CO
2
, H
2
, Fe
2+
[mM]
Fa y Acids [mM]
R
ESULTS
63
3.2.
Iden i ica ion o Ac i e P oka yo es in [
13
C]-Cellulose,
-Cellobiose, and -Glucose Supplemen ed T ea men s by RNA
S able Iso ope P obing
S able iso ope p obing was used o iden i y p oka yo ic soil o ganisms ha
di ec ly inco po a ed [
13
C]-ca bon in o hei RNA by assimila ing
[
13
C]-cellulose, -cellobiose, o -glucose unde oxic and anoxic condi ions (2.3.1, 3.1).
Al hough he isk o c oss- eeding by [
13
C]-ca bon dioxide was minimized (2.3.1) i
canno be excluded ha some soil o ganisms inco po a ed [
13
C]-ca bon by
assimila ion o [
13
C]-b eakdown p oduc s (e.g., a y acids) du ing p olonged
incuba ion. The e o e, RFLP analysis was no only pe o med a he end o
incuba ion bu also in be ween o de ec ime-dependen shi s in he ac i e mic obial
communi y (Figu e 11).
3.2.1. Dis ibu ion o RNA in G adien F ac ions and Selec ion o ’Ligh ’ and
’Hea y’ F ac ions o Molecula Analyses
RNA ex ac ed om [
12
C]- and [
13
C]-cellulose, -cellobiose, o -glucose
ea men s was subjec ed o isopycnic cen i uga ion in a densi y g adien o sepa a e
‘hea y’ [
13
C]-RNA o saccha ide-assimila ing, i.e., labeled, o ganisms om ‘ligh ’
[
12
C]-RNA o non-labeled o ganisms. A RNA- ee g adien (blank) was used o
de e mine he buoyan densi y o any ac ion (Figu e 12).
The buoyan densi y o each ac ion dec eased linea ly and g adien s we e
ep oducible and compa able be ween h ee independen cen i uga ion uns
(Figu e 12). Buoyan densi ies anged om 1.84 ± 0.01 g ml
-1
o 1.76 ± 0.01 g ml
-1
.
Since he g adien s we e highly ep oducible (Figu e 12) he dis ibu ion o RNA in
g adien ac ions was only de e mined exempla ily o one g adien (Figu e 13).
R
ESULTS
64
Figu e 12. Buoyan densi y o g adien solu ion in ac ions o blank g adien s
ha lack RNA a 25°C.
Closed symbols: cen i uga ion un 1; g ey symbols: cen i uga ion
un 2; open symbols: cen i uga ion un 3. Lines ep esen linea eg essions (R
2
= 0.98).
E o ba s indica e s anda d de ia ions (n = 10).
RNA was dis ibu ed o e he whole g adien wi h highes concen a ions in
ac ions numbe ou and se en, indica ing a pa ial sepa a ion o [
13
C]- and [
12
C]-
RNA (Figu e 12). Full sepa a ion o ‘hea y’ and ‘ligh ’ RNA de i ed om pu e cul u es
is possible (Lüde s e al. 2004), bu no likely o en i onmen al samples. Based on
li e a u e (Lüde s e al. 2004; Mane ield e al. 2002a) wo ac ions ha p esumably
ep esen ed labeled ‘hea y’ o unlabeled ‘ligh ’ RNA we e selec ed o u he
analyses. F ac ions numbe h ee and ou (buoyan densi ies o 1.813 ± 0.00 –
1.821 ± 0.01 g ml
-1
) we e pooled and co espond o ‘hea y’ RNA. F ac ions numbe
eigh and nine (buoyan densi ies o 1.767 ± 0.00 – 1.776 ± 0.00 g ml
-1
) we e pooled
o analyses o unlabeled ‘ligh ’ RNA (Figu e 13). RNA o ‘hea y’ and ‘ligh ’ ac ions
o [
12
C]- and [
13
C]- ea men s was ansc ibed in o cDNA and used o compa a i e
RFLP analyses o bac e ial and a chaeal 16S RNA cDNA sequences.
F ac ion
1 2 3 4 5 6 7 8 9 10
Buyoan Densi y [g ml-1]
1.74
1.76
1.78
1.80
1.82
1.84
1.86
R
ESULTS
65
Figu e 13. Dis ibu ion o RNA in g adien ac ions o anoxic [
13
C]-cellobiose
ea men s a e 24 days o incuba ion
. Red indica es ac ions ha we e chosen o
analyses o ‘hea y‘ RNA. Blue indica es ac ions ha we e chosen o analyses o ‘ligh ‘
RNA. E o ba s indica he s anda d de ia ion (n = 3). Buoyan densi y o ‘ligh ’ ac ions was
1.767 ± 0.00 – 1.776 ± 0.00 g ml
-1
. Buoyan densi y o ‘hea y’ ac ions was 1.813 ± 0.00 –
1.821 ± 0.01 g ml
-1
.
3.2.2. TRFLP P o iles o A chaeal 16S RNA cDNA Sequences
A chaeal 16S RNA cDNA genes we e diges ed wi h TaqI, esul ing in wo
majo RFs (142 bp, 154 bp) and some mino RFs wi h ela i e luo escence alues
below 5% (Figu e 14). These RFs we e p esen in each sample o cellulose-,
cellobiose-, o glucose-supplemen ed ea men s (da a no shown). Nei he
di e ences be ween [
13
C]- and [
12
C]- ea men s no be ween ‘ligh ’ and ‘hea y’
ac ions o [
13
C]- ea men s we e de ec ed (Figu e 14). This gene al esul sugges ed
ha mesophilic A chaea we e o mino o no consequence o he p ima y
consump ion o supplemen al subs a es. A chaeal 16S RNA cDNA genes we e no
used o cloning, bu in silico analyses e ealed ha he RFs migh ep esen
species o Me hanoco pusculaceae, Me hanococcaceae, and Me hanobac e iaceae
( RF 142 bp). TRF 154 bp could no be assigned o known sequences.
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
3
4
8
9
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
3
4
8
9
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
33
44
88
99
RNA [ng µl
-1
]
0.0 0.2 0.4 0.6
F ac ion
1
2
3
4
5
6
7
8
9
10
33
44
88
99
R
ESULTS
66
Figu e 14. TRFLP gel image o a chaeal 16S RNA cDNA genes
. Res ic ion
enzyme was TaqI. S, leng h s anda d 75 – 1,000 bp; LF, ‘ligh ‘ ac ion; SF, ‘hea y‘ ac ion;
Rep., Replica es; X, Lane was no analysed.
900
800
700
600
500
450
400
350
300
250
100
75
200
150
900
800
700
600
500
450
400
350
300
250
100
75
200
150
1000
13
LF
13
SF
13
LF
13
SF
12
SF
12
LF
12
SF
12
LF
202024 240204 24
3 Rep.
0
612
3 Rep.
0 204 24
3 Rep.
612
3 Rep.
0126 126
S S
S
Day
XX
900
800
700
600
500
450
400
350
300
250
100
75
200
150
900
800
700
600
500
450
400
350
300
250
100
75
200
150
900
800
700
600
500
450
400
350
300
250
100
75
200
150
1000
900
800
700
600
500
450
400
350
300
250
100
75
200
150
1000
13
LF
13
SF
13
LF
13
SF
12
SF
12
LF
12
SF
12
LF
202024 240204 24
3 Rep.
0
612
3 Rep.
0 204 24
3 Rep.
612
3 Rep.
0126 126
S S
S
Day
XX XX
R
ESULTS
67
3.2.3. TRFLP P o iles o Bac e ial 16S RNA cDNA Sequences
P ima ily, RF p o iles analysed o he las ime poin s we e compa ed, i.e., o
each ea men ‘ligh ’ and ‘hea y’ ac ion o oxic and anoxic ea men s
supplemen ed wi h ei he [
13
C]-cellulose, [
13
C]-cellobiose, [
13
C]-glucose, [
12
C]-
cellulose, [
12
C]-cellobiose, o [
12
C]-glucose we e compa ed (Figu e 15 and Figu e
16). Sepa a ion o [
12
C]- and [
13
C]-RNA du ing cen i uga ion should be e lec ed in
di e en RF pa e ns be ween ‘ligh ’ and ‘hea y’ ac ions.
3.2.3.1. Compa ison o ‘Hea y’ and ‘Ligh ’ F ac ions in Oxic and Anoxic [
12
C]- o
[
13
C]-T ea men s
Res ic ion diges ion o bac e ial 16S RNA cDNA sequences de i ed om [
13
C]-
ea men s esul ed in di e en RF pa e n o he ‘ligh ’ and ‘hea y’ ac ions (Figu e
15), con i ming inco po a ion o [
13
C]-ca bon in o RNA by pa s o he mic obial
communi y and success ul sepa a ion o [
12
C]- and [
13
C]-RNA in he g adien solu ion.
Assuming ha labeled axa a e p esen ed by (i) e e y RF ha is exclusi ely p esen
in he ‘hea y’ ac ion bu no in he ‘ligh ’ ac ion and (ii) e e y RF ha exhibi s a
highe ela i e luo escence in he ‘hea y’ han in he ‘ligh ’ ac ion, 31 RFs (in o al)
we e pu a i ely labeled in [
13
C]- ea men s a he end o incuba ion.
R
ESULTS
68
Figu e 15. TRFLP pa e n o bac e ial 16S RNA cDNA sequences in ‘ligh ‘
( ac ion 8+9: 1.767-1.776 g ml
-1
) and ‘hea y‘ ac ions ( ac ion 3+4: 1.181-1.183
g ml
-1
) o [
13
C]-supplemen ed ea men s a he la es analysed ime poin .
(A)
Cellulose ea men a e 70 days. (B) Cellobiose ea men a e 12 days (oxic) and 24 days
(anoxic). (C) Glucose ea men a e 12 days (oxic) and 24 days (anoxic). Values a e he
mean o iplica es ([
13
C]-cellobiose, [
13
C]-glucose) o duplica es ([
13
C]-cellulose). E o ba s
indica e s anda d de ia ions. Ba s ha ep esen pu a i ely labeled RFs (i.e., RFs
exclusi ely occu ed in ’hea y‘ ac ions o a e highe in ela i e luo escence han he
co esponding RFs in ‘ligh ‘ ac ions) a e ma ked wi h an as e isk.
B
A
**
*
*
**
*
*
*
****
*
*
*
*
*
**
T-RF Leng h [bp]
C
*******
*
*
*
*
60
30
0
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
60
30
60
30
60
30
100
50
0
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
Rela i e Fluo escence [%]
100
50
100
50
100
50
80
40
0
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
80
40
80
40
80
40
R
ESULTS
69
Compa isons o ‘ligh ’ and ‘hea y’ ac ions may lead o an o e es ima ion o he
ac i e pa o he mic obial communi y, since RFs in he ‘hea y’ ac ion do no
necessa ily esul om labeled RNA, bu can esul om co-mig a ed unlabeled RNA
(Lüde s e al. 2004). The p esence o unlabeled RFs in ‘hea y’ ac ions becomes
appa en by compa ison o ‘hea y’ and ‘ligh ’ ac ions o [
12
C]- ea men s (Figu e 16)
ha ne e ecei ed [
13
C]-ca bon. In o al, eigh RFs we e exclusi ely p esen in
‘hea y’ ac ions o [
12
C]- ea men s, al hough a [
13
C]-labeling was impossible. These
unlabeled geno ypes in ‘hea y’ ac ions could be ela ed o sequences ha had high
G+C con en s (53 – 61%, 3.2.4.2.2). They may ha e hus been un olded RNA
molecules yielding buoyan densi ies simila o labeled geno ypes (Lüde s e al.
2004). To a oid his bias iden i ica ion o labeled RFs was based on he compa ison
o ‘hea y’ ac ions o [
12
C]- ea men s wi h ‘hea y’ ac ions o [
13
C]- ea men s
(3.2.3.2).
R
ESULTS
70
Figu e 16. TRFLP pa e n o bac e ial 16S RNA cDNA sequences in ‘ligh ‘
( ac ion 8+9: 1.767-1.776 g ml
-1
) and ‘hea y‘ ac ions ( ac ion 3+4: 1.181-1.183
g ml
-1
) o [
12
C]-supplemen ed ea men s a he la es analysed ime poin .
(A)
Cellulose ea men a e 70 days. (B) Cellobiose ea men a e 12 days (oxic) and 24 days
(anoxic). (C) Glucose ea men a e 12 days (oxic) and 24 days (anoxic). Fluo escence
alues we e no malized agains he lowes single luo escence; alues below 5% we e
excluded om he analysis. Ba s ha ep esen RFs ha exclusi ely occu in ’hea y‘
ac ions a e ma ked wi h an as e isk.
**
*
*
***
*
60
30
0
60
30
60
30
60
30
80
40
0
Rela i e Fluo escence [%]
80
40
80
40
80
40
80
540
0
80
40
80
40
80
40
T-RF Leng h [bp]
69
91
143
153
163
172
205
279
288
295
325
433
438
447
456
469
474
480
486
490
494
497
500
507
511
520
523
526
540
547
552
603
772
891
903
920
80
134
149
156
164
201
208
283
292
298
327
436
443
452
466
473
476
483
488
492
496
498
503
508
514
522
525
534
542
549
559
690
870
899
912
B
A
C Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
**
*
*
***
*
60
30
0
60
30
60
30
60
30
80
40
0
Rela i e Fluo escence [%]
80
40
80
40
80
40
80
540
0
80
40
80
40
80
40
T-RF Leng h [bp]
69
91
143
153
163
172
205
279
288
295
325
433
438
447
456
469
474
480
486
490
494
497
500
507
511
520
523
526
540
547
552
603
772
891
903
920
80
134
149
156
164
201
208
283
292
298
327
436
443
452
466
473
476
483
488
492
496
498
503
508
514
522
525
534
542
549
559
690
870
899
912
80
134
149
156
164
201
208
283
292
298
327
436
443
452
466
473
476
483
488
492
496
498
503
508
514
522
525
534
542
549
559
690
870
899
912
B
A
C Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
Oxic ‘ligh ‘
Oxic ‘hea y‘
Anoxic ‘hea y‘
Anoxic ‘ligh ‘
R
ESULTS
71
3.2.3.2. Iden i ica ion o Labeled RFs by Compa ison o ‘Hea y’ F ac ions
be ween [
12
C]- and [
13
C]-T ea men s and Phylogene ic A ilia ion
A conse a i e app oach was pe o med o iden i y labeled RFs (3.2.3.1). TRFs
we e sco ed as labeled when hey we e only p esen in [
13
C]-p o iles (e.g., RF
507 bp, Figu e 17) o when he ela i e in ensi y in [
13
C]-p o iles was signi ican ly
highe han in [
12
C]-p o iles a he same espec i e ime in e al (e.g., RF 490 bp,
Figu e 17). Labeled RFs we e assigned o amily-le el axa based on gene lib a y
analyses (Table 19 – Table 22).
3.2.3.2.1. Bac e ia ha Inco po a ed [
13
C]-Ca bon in Oxic T ea men s
[
13
C]-cellulose supplemen a ion esul ed in wel e labeled RFs o which RFs
438, 466, 490, 507, 525, and 540 bp we e obse ed a days 35 and 70, whe eas
RFs 80, 443, 456, 473, 497, and 503 bp we e only de ec ed a day 70 (Figu e 17).
No RF was dominan o con ibu ed o mo e han 30% o he o al luo escence wha
co ela ed wi h he equency o co esponding labeled geno ypes ob ained om
gene lib a ies om oxic cellulose-supplemen ed ea men s (Table 22, 3.2.4.2.1). Ten
RFs we e iden i ied as known and new amily-le el axa wi hin he phyla
Ac inobac e ia, Alphap o eobac e ia, Bac e oide es, Be ap o eobac e ia, Chlo o lexi,
Del ap o eobac e ia, Fi micu es, Gammap o eobac e ia, Planc omyce es, and wo
deep-b anching g oups wi hin Bac e ia (‘Bac2’) and P o eobac e ia (‘P o 1’) (Table
19). Two RFs (503 bp, 540 bp) could no be iden i ied.
R
ESULTS
78
[
13
C]-cellobiose supplemen a ion esul ed in h ee labeled RFs o which RF
523 bp was p esen a day 4, 20, and 24, and exhibi ed he highes ela i e
luo escence (Figu e 21). TRF 494 bp was only de ec ed a day 4, RF 903 a day 4
and 20, bu disappea ed a day 24 (Figu e 21). The mos dominan RF 523 bp
a ilia ed wi h he phylum Fi micu es (Table 20 and Table 22) wha co esponds o he
p e alence o his phylum in gene lib a ies (Table 22). TRF 494 bp ep esen ed
membe s o he phyla Gammap o eobac e ia and Del ap o eobac e ia, axa o mino
impo ance in bo h ela i e luo escence and equencies in gene lib a ies (Table 20
and Table 22). TRF 903 bp could no be iden i ied (Table 20).
Figu e 21. TRFLP pa e n o bac e ial 16S RNA cDNA sequences in ‘hea y’
ac ions ( ac ion 3+4: 1.181-1.183 g ml
-1
) o cellobiose-supplemen ed anoxic
ea men s
. (A) [
13
C]-cellobiose ea men a e 0, 4, 10, and 24 days. (B) [
12
C]-cellobiose
ea men a e 20 and 24 days. Time poin s we e chosen conce ning changes in p ocesses
du ing subs a e u iliza ion (Figu e 11). The [
12
C]-g adien o day 0 and 4 was no gene a ed.
Fluo escence alues we e no malized agains he lowes single luo escence; alues below
5% we e excluded om he analysis. E o ba s indica e s anda d de ia ions. [
13
C]-labeled
RFs a e indica ed by hei leng h and ma ked by an a ow (494 bp; 523 bp; 903 bp).
A [
13
C]-Cellobiose (Anoxic)
Rela i e
Fluo escence [%]
0 days
20 days
24 days
B [
12
C]-Cellobiose (Anoxic)
4 days
20 days
Rela i e
Fluo escence [%]
RF Leng h [bp]
70 160 480 920
24 days
RF Leng h [bp]
494bp
➤
70 160 480 920
523bp
➤
903bp
➤
75
50
25
523bp
➤
903bp
➤
523bp
➤
523bp
➤
523bp
➤
903bp
➤
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
A [
13
C]-Cellobiose (Anoxic)
Rela i e
Fluo escence [%]
0 days
20 days
24 days
B [
12
C]-Cellobiose (Anoxic)
4 days
20 days
Rela i e
Fluo escence [%]
RF Leng h [bp]
70 160 480 920
24 days
RF Leng h [bp]
494bp
➤
70 160 480 920
523bp
➤
903bp
➤
75
50
25
75
50
25
523bp
➤
903bp
➤
523bp
➤
523bp
➤
523bp
➤
903bp
➤
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
75
50
25
R
ESULTS
79
[
13
C]-glucose supplemen a ion esul ed in h ee labeled RFs (Figu e 22) o
which only RF 520 bp was also p esen in [
12
C]- ea men s. TRF 508 and 520 bp
we e only labeled a day 10 and 24, whe eas RF 496 was labeled a all ime poin s
(Figu e 22). TRF 520 bp was he dominan labeled RF and a ilia ed wi h he phylum
Fi micu es which also ep esen ed he mos equen axon in he co esponding
gene lib a y (Table 20 and Table 22). TRF 496 bp was assigned o he phylum
Gammap o eobac e ia (Table 20) which was a mino axon in gene lib a ies (Table
22). TRF 508 bp could no be iden i ied, al hough i exhibi ed a ela i e luo escence
o abou 10%.
Figu e 22. TRFLP pa e n o bac e ial 16S RNA cDNA sequences in ‘hea y’
ac ions ( ac ion 3+4: 1.181-1.183 g ml
-1
) o glucose-supplemen ed anoxic
ea men s
. (A) [
13
C]-glucose ea men a e 0, 4, 10 and 24 days. (B) [
12
C]-glucose
ea men a e 0, 4, and 24 days. Time poin s we e chosen conce ning changes in
p ocesses du ing subs a e u iliza ion (Figu e 11). The [
12
C]-g adien o day 10 was no
gene a ed. Fluo escence alues we e no malized agains he lowes single luo escence;
alues below 5 % we e excluded om he analysis. E o ba s indica e s anda d de ia ions.
[
13
C]-Labeled RFs a e indica ed by hei leng h and ma ked by an a ow (496 bp; 508 bp;
520 bp).
A [
13
C]-Glucose (anoxic)
Rela i e Fluo escence [%]
0 days
0 days
4 days
24 days
B [
12
C]-Glucose (anoxic)
4 days
10 days
24 days
Rela i e Fluo escence [%]
RF Leng h [bp]
70 290 480 540
70 290 480 540
RF Leng h [bp]
496bp
508bp
520bp
➤
➤
➤
520bp
➤
520bp
➤
520bp
➤
520bp
➤
496bp
➤
496bp
➤
508bp
➤
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
A [
13
C]-Glucose (anoxic)
Rela i e Fluo escence [%]
0 days
0 days
4 days
24 days
B [
12
C]-Glucose (anoxic)
4 days
10 days
24 days
Rela i e Fluo escence [%]
RF Leng h [bp]
70 290 480 540
70 290 480 540
RF Leng h [bp]
496bp
508bp
520bp
➤
➤
➤
520bp
➤
520bp
➤
520bp
➤
520bp
➤
496bp
➤
496bp
➤
508bp
➤
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
60
40
20
R
ESULTS
80
TRFLP analyses and 16S RNA cDNA gene lib a ies o anoxic [
13
C]- ea men s
showed ha he phylum Fi micu es was p e alen in all ea men s, i.e., cellulose-,
cellobiose-, and glucose-supplemen ed ea men s (Table 22). Almos all labeled
geno ypes o he phylum Fi micu es we e ela ed o he amily Clos idiaceae.
Di e en sub-g oups wi hin his amily we e selec i ely ac i a ed in ega d o he ype
o subs a e. Sequences o Clus e III Clos idiaceae we e closely ela ed (> 97%
sequence simila i y) o he s ic anae obic celluloly ic species Clos idium
celluloly icum and C. he mocellum (Magnusson e al. 2009; Pe i demange e al.
1984; Zhang and Lynd 2005), whe eas sequences de i ed om [
13
C]-cellobiose and
–glucose ea men s we e closely ela ed (> 98% simila i y) o he saccha oly ic
species C. bu y icum and C. incen ii (Figu e A2; Moun o e al. 1997; Ske man e
al. 1980). An unclassi ied clus e (‘Cellu1’) wi hin he phylum Bac e oide es and he
amily Kineospo iaceae (phylum Ac inobac e ia) we e majo ac i e axa in cellulose-
supplemen ed ea men s (Table 22, Figu e A1 – Figu e A3), bu no in cellobiose- o
glucose-supplemen ed ea men s. The lack o hese axa in cellobiose and glucose-
supplemen ed ea men s (Table 22) indica es ha labeling by c oss- eeding on
cellulose-de i ed cellodex ins was unlikely.
Table 20. Iden i ica ion and occu ence o labeled RFs in anoxic [
13
C]-
ea men s.
T ea men
RF [bp] Iden i y [phylum: amily]
a
Label
b
[day]
Cellulose-supplemen ed
Anoxic 143 Ac inobac e ia: In aspo angiaceae,
Kineospo iaceae, S ep omyce aceae
Del ap o eobac e ia: Geobac e aceae
70
149 Ac inobac e ia: Cellulomonadaceae
Alphap o eobac e iaceae: Hyphomic obiaceae
70
205 Bac e oide es: ‘Cellu1’
c
Bac e ia: ‘Bac1’
c
, ‘Bac3’
c
Fi micu es: Clos idiaceae, Paenibacillaceae,
‘Clos1’
c
35, 70
490 Be ap o eobac e ia: Rhodocyclaceae
Del ap o eobac e ia: Pelobac e aceae
35
522 Fi micu es: Clos idiaceae, ‘Clos2’
c
35, 70
540 Bac e oide es: ‘Cellu2’
c
, ‘Cellu3’
c
35
899 n.i. 35
R
ESULTS
81
T ea men
RF [bp] Iden i y [phylum: amily]
a
Label
b
[day]
Cellobiose
-
supplemen ed
Anoxic 494 Gammap o eobac e ia: Ae omonadaceae,
‘Gam1’
c
Del ap o eobac e ia: Pelobac e aceae
4
523 Fi micu es: Clos idiaceae, ‘Clos4’
c
4, 20, 24
903 n.i. 4, 20
Glucose
-
supplemen ed
Anoxic 496 Gammap o eobac e ia: Ae omonadaceae,
En e obac e iaceae 4, 10, 24
508 n.i. 10, 24
520 Fi micu es: Clos idiaceae 10, 24
n.i., No iden i ied.
a
Based on expe imen al gene lib a ies.
b
Fi s labeling a no ed day o incuba ions in ‘hea y’ RNA ac ions.
c
New amily-le el axon based on 16S RNA gene simila i ies < 87% o nex cul i a ed
species.
3.2.4. Bac e ial 16S RNA cDNA Gene Lib a ies o ‘Hea y’ F ac ions
To iden i y RFs ela ed o labeled axa, six 16S RNA cDNA gene lib a ies we e
cons uc ed, i.e., 16S RNA cDNA genes de i ed om ‘hea y’ ac ions o oxic and
anoxic [
13
C]-cellulose, -cellobiose, o -glucose ea men s we e used o cloning.
3.2.4.1. Ra e ac ion and Co e age
In o al, 828 sequences (> 350 bp leng h) we e analysed. The numbe o clones
pe lib a y anged be ween 104 and 177 (Table 21). The co e age was abo e 90% in
i e o six lib a ies, indica ing ha he majo di e si y on amily-le el (> 87%
sequence simila i y) was co e ed by he analyses (Table 21). Some lib a ies
exhibi ed a high numbe o sequences ha could no be assigned o labeled RFs.
These sequences we e de ined o ep esen unlabeled bac e ial axa (Table 23),
pa ly esul ing in a low p opo ion o labeled clones in ‘hea y’ ac ions (Table 21).
R
ESULTS
82
Table 21. Cha ac e is ics o 16S RNA cDNA gene lib a ies o ’hea y’ ac ions
o oxic and anoxic [
13
C]-cellulose, [
13
C]-cellobiose, and [
13
C]-glucose
ea men s.
Numbe o
clones pe
lib a y
Numbe o
amily-le el
OTUs
Labeled
clones
[%]
Unlabeled
clones
[%] Co e age
[%]
Cellulose-supplemen ed
Oxic 163 93 23.3 76.7 64.4
Anoxic 177 27 89.8 10.2 93.8
Cellobiose-supplemen ed
Oxic 122 19 45.2 54.8 93.4
Anoxic 130 17 64.6 35.4 94.7
Glucose-supplemen ed
Oxic 104 15 63.6 36.4 96.2
Anoxic 132 28 23.3 76.7 90.9
Ra e ac ion analyses suppo ed he abo e men ioned indings (i.e., de ec ion o
he majo di e si y), as indica ed by he ou -pla eauing a e ac ion cu es (Figu e 23).
The a e ac ion cu e o oxic cellulose-supplemen ed ea men s was s eep (Figu e
23 A) wha co esponded o he low co e age o 64% o his gene lib a y (Table 21).
Gene lib a ies o oxic and anoxic cellobiose- and glucose-supplemen ed ea men s
showed bo h a good co e age (Table 21), and a e ac ion cu es nea ly ou -
pla eaued (Figu e 23 B and C). In gene al, di e ences in he di e si y wi hin all
ea men s we e obse ed. Mo e amily-le el OTUs we e de ec ed in oxic cellulose-
and oxic cellobiose-supplemen ed ea men s han in he co esponding anoxic
ea men s, whe eas oxic glucose-supplemen ed ea men s showed less OTUs han
he co esponding anoxic ea men s (Table 21). The o al numbe o de ec ed OTUs
was highes in cellulose-supplemen ed ea men s.
R
ESULTS
83
Figu e 23. Ra e ac ion cu es o 16S RNA cDNA gene sequences o ‘hea y‘
ac ions o oxic and anoxic [
13
C]- ea men s
. (A) Cellulose-supplemen ed; (B)
Cellobiose-supplemen ed; (C) Glucose-supplemen ed. , oxic ea men s; , anoxic
ea men s. Solid lines indica e he 95% con idence in e als.
0
25
50
75
100
A
Numbe o OTUs
0
5
10
15
20
B
Numbe o Clones
0 25 50 75 100 125 150 175
0
10
20
30
C
0
25
50
75
100
A
Numbe o OTUs
0
5
10
15
20
B
Numbe o Clones
0 25 50 75 100 125 150 175
0
10
20
30
C
R
ESULTS
84
3.2.4.2. G ouping o Sequences in o Family-le el OTUs and hei Phylogene ic
A ilia ion
16S RNA cDNA gene sequences we e g ouped in o amily-le el OTUs i
sequence simila i y was highe han 87 % (Ya za e al. 2008). TRFLP analysis was
used o di e en ia e be ween geno ypes o labeled and non-labeled axa de ec ed in
16S RNA cDNA gene lib a ies.
3.2.4.2.1. Labeled Taxa
Fou y-eigh amily-le el axa wi hin en bac e ial phyla we e iden i ied as
o ganisms ha assimila ed ca bon de i ed om [
13
C]-cellulose, -cellobiose,
o -glucose. Twen y-eigh o hese axa did no closely a ilia e wi h known amilies
(Table 22, Figu e A1 - Figu e A3). Ac i e axa di e ed in ega d o he subs a e ype
and he a ailabili y o O
2
, indica ing ha (i) cellulose supplemen a ion selec ed o
o he axa han cellobiose and glucose supplemen a ion did, and (ii) oxic condi ions
selec ed o o he axa han anoxic condi ions did (Table 22). Taxa ha showed high
ela i e abundances in gene lib a ies o oxic glucose- and cellobiose-supplemen ed
ea men s we e he amilies In aspo angiaceae (5 – 17%) and Mic ococcaceae
(30%) o he phylum Ac inobac e ia (Table 22, Figu e A1). In con as , majo ac i e
axa in anoxic glucose- and cellobiose-supplemen ed ea men s we e closely ela ed
o saccha oly ic Clos idiaceae (Clus e I Clos idiaceae) o he phylum Fi micu es
(19 – 53%) (Table 22, Figu e A2). Planc omyce aceae (Planc omyce es), a deep-
b anching new amily-le el axon (‘Deha1’) o he phylum Chlo o lexi, and ou new
axa (‘Sphingo1-4’) o Sphingobac e iales (Bac e oide es) we e he mos abundan
g oups in gene lib a ies o oxic cellulose-supplemen ed ea men s and exhibi ed
ela i e abundances o almos 4%, 5%, and 3%, espec i ely (Table 22, Figu e A3).
Kineospo iaceae (Ac inobac e ia), celluloly ic Clos idiaceae (Clus e III
Clos idiaceae, phylum Fi micu es), and h ee new deep-b anching amily-le el axa
(‘Cellu1-3’) o he phylum Bac e oide es we e dominan in gene lib a ies o anoxic
cellulose-supplemen ed ea men s and had ela i e abundances o abou 16%, 28%,
and 22%, espec i ely (Table 22, Figu e A1 – Figu e A3). These g oups we e
analysed o hei esponse o changing a ailabili ies o O
2
and pes icides in u he
expe imen s (3.6; 3.5).
R
ESULTS
85
Table 22. Rela i e abundances o labeled OTUs ob ained om 16S RNA cDNA
gene lib a ies om ‘hea y’ ac ions o [
13
C]- ea men s and hei phylogene ic
a ilia ion.
Rela i e Abundances [%]
Phyla and amilies OTUs
a
Cellulose Cellobiose Glucose
oxic
anoxic
oxic
anoxic
oxic
anoxic
Ac inobac e ia
Cellulomonadaceae 22, 23, 78 1.23
0.57 0.81
- - -
In aspo angiaceae 5, 60, 79 0.61
1.14 4.84
- 16.8
-
Kineospo iaceae 2, 46 - 21.7 4.03
- 5.61
-
Mic ococcaceae 3, 18, 42,
54, 57, 58,
59, 61, 80
- - 29.8
- 29.9
-
Mycobac e iaceae 56, 62 - - 2.24
- - -
Nakamu ellaceae 55 - - - - 0.93
-
Noca diaceae 44, 65 - - 1.61
- - -
Noca dioidaceae 13, 47, 63 0.61
- - - 4.67
-
S ep omyce aceae 11 - 2.29 - - - -
‘Mic o1’
(Mic ococcineae)
b
34 0.61
- - - - -
‘Mic o2’
(Mic ococcineae)
b
41 - - 0.81
- - -
‘Mic o3’
(Mic ococcineae)
b
43 - - - - 0.93
-
‘Mic o4’
(Mic ococcineae)
b
48 0.61
- - - - -
‘Ac ino1’
(Ac inomyce ales)
b
40 0.61
- - - - -
Alphap o eobac e ia
Hyphomic obiaceae 72 - 0.57 - - - -
‘Rhizo1’ (Rhizobiales)
b
75 0.61
- - - - -
‘Rhizo2’ (Rhizobiales)
b
76 0.61
- - - - -
R
ESULTS
86
Rela i e Abundances [%]
Phyla and amilies OTUs
a
Cellulose Cellobiose Glucose
oxic
anoxic
oxic
anoxic
oxic
anoxic
Bac e oide es
‘Sphingo1’
(Sphingobac e iales)
b
28 1.23
- - - - -
‘Sphingo2’
(Sphingobac e iales)
b
36 0.61
- - - - -
‘Sphingo3’
(Sphingobac e iales)
b
38 0.61
- - - - -
‘Sphingo4’
(Sphingobac e iales)
b
50 0.61
- - - - -
‘Cellu1’
c
6 - 13.7 - - - -
‘Cellu2’
c
14 - 1.71 - - - -
‘Cellu3’
c
74 - 0.57 - - - -
Be ap o eobac e ia
Ni osomonadaceae 67 - - - - 0.93
-
Oxalobac e aceae 66 0.61
- - - - -
Rhodocyclaceae 7 - 4.00 - - - 0.75
‘Bu k1’
(Bu kholde iales)
b
24 - - - - 0.93
-
Chlo o lexi
‘Deha1’
(Dehalococcoide es)
b
8 4.91
- - - - -
Del ap o eobac e ia
Geobac e aceae 27, 35 - 0.57 - - 0.93
-
Pelobac e aceae 12 - 1.14 - 0.77 - -
‘Desu1’
(Desul u omonadales)
b
19 1.23
- - - - -
‘Myxo1’
(Myxococcales)
b
31 0.61
- - - - -
R
ESULTS
87
Rela i e Abundances [%]
Phyla and amilies OTUs
a
Cellulose Cellobiose Glucose
oxic
anoxic
oxic
anoxic
oxic
anoxic
Fi micu es
Clos idiaceae
1, 4, 16, 20,
21, 25, 51,
52, 53, 68
- 28.0
- 53.1 - 18.8
Paenibacillaceae 15 - 0.57 - - - -
‘Clos1’ (Clos idiales)
b
29 - 1.14 - - - -
‘Clos2’ (Clos idiales)
b
37 - 0.57 - - - -
‘Clos3’ (Clos idiales)
b
49 0.61
- - - - -
‘Clos4’ (Clos idiales)
b
82 - - - 0.77 - -
Gammap o eobac e ia
Ae omonadaceae 70, 73 - - - 0.77 - 2.26
En e obac e iaceae 9 - - - - - 1.50
‘Ch om1’
(Ch oma iales)
b
26 0.61
- - - - -
‘Gam1’
e
81 - - - 0.77 - -
Planc omyce es
Planc omyce aceae 10, 32, 33 3.68
- - - - -
P o eobac e ia
‘P o 1’
77 0.61
- - - - -
Bac e ia
‘Bac1’
g
17 - 1.71 - - - -
‘Bac2’
h
30 0.61
- - - - -
‘Bac3’
i
64 - 1.14 - - - -
-, No de ec ed.
a
Iden i ica ion o OTUs by BLASTn (h p://blas .ncbi.nlm.nih.go /).
b
New amily-le el axon based on 16S RNA gene simila i ies < 87% o nex cul i a ed
species (Ya za e al. 2008).
c
Nex cul i a ed species: P olixibac e bellanii o ans (AY918928; 86% 16S RNA gene
simila i y).
d
Sequences de i ed om cellulose-supplemen ed ea men s clus e ed o Clus e III
Clos idiaceae, sequences om cellobiose- and glucose-supplemen ed ea men s clus e ed
o Clus e I Clos idiaceae (acco ding o Collins e al. 1994).
R
ESULTS
94
RNA cDNA gene lib a ies o ‘hea y’ ac ion bu no iden i ied as labeled axon
(3.2.4.2.2). Cloning o qPCR p oduc s ampli ied by p ime s speci ic o celluloly ic
Clos idiaceae (Clus e III Clos idiaceae) e ealed ha none o he analysed
sequences was ela ed o sequences o his axon (Table 26). In con as o pu e
a ge DNA and non- a ge DNA o which he assay was speci ically wo king, i was
no possible o ampli y any a ge sequence in en i onmen al samples. Assays
designed o quan i ica ion o he new amily-le el axa ‘Cellu1-3’, ‘Sphingo1-4’ and
‘Deha1’ yielded 96%, 61%, and 80% (Table 26) speci ici y, espec i ely. Non- a ge
sequences ha we e ampli ied by hese assays we e ela ed o a ious o ganisms in
he phyla Cyanobac e ia, Del ap o eobac e ia, Fi micu es, Gemma imonade es, and
Ni ospi ae.
Al hough published p o ocols we e used o he quan i ica ion o o al Bac e ia
and A chaea, he assays we e checked o hei speci ici y. The Bac e ia- a ge ing
p ime s we e highly speci ic and did no yield any non- a ge axon (i.e., sequences
we e no non-bac e ial) (Table 26). In con as , app oxima ely 44% o he sequences
ha we e ampli ied wi h he A chaea- a ge ing p ime s we e non-a chaeal (i.e.,
sequences we e bac e ial).
R
ESULTS
95
Table 26. Speci ici y o qPCR assays.
Ta ge
sequences
[%]
a
Non- a ge
sequences
[%]
a
A ilia ion o non-
a ge s
(phylum, numbe o
sequences)
Family
-
le el
a
ssays
1 Mic ococcaceae &
Cellulomonadaceae 100.0 (19) 0.0 (0) ---
2 Kineospo iaceae &
Noca dioidaceae 78.9 (15) 21.1 (4) Acidobac e ia, 1
Cyanobac e ia, 2
Fi micu es, 1
3 Clus e I Clos idiaceae 84.2 (16) 15.8 (3) Gammap o eobac e ia,
3
4 Clus e III Clos idiaceae 0.0 (0) 100.0 (20) Acidobac e ia, 6
Ac inobac e ia, 1
Alphap o eobac e ia, 6
Del ap o eobac e ia, 5
Fi micu es, 2
5 Planc omyce aceae 100.0 (20) 0.0 (0) ---
6 ‘Cellu1-3’
b
(Bac e oide es) 95.5 (21) 4.5 (1) Cyanobac e ia, 1
7 ‘Sphingo1-4’
b
(Bac e oide es) 61.1 (11) 38.9 (7) Del ap o eobac e ia, 3
Gemma imonade es, 4
8 ‘Deha1’
b
(Chlo o lexi) 80.0 (16) 20.0 (4) Ni ospi ae, 2
Fi micu es, 2
Domain
-
le el
a
ssays
9 Bac e ia 100.0 (20) 0.0 (0) ---
10
A chaea 55.6 (10) 44.4 (8) Ac inobac e ia, 1
Alphap o eobac e ia, 1
Bac e oide es, 1
Be ap o eobac e ia, 2
Fi micu es, 1
Planc omyce es, 2
a
Pa en he ical alues a e he numbe o sequences.
b
Name and a ilia ion as de ined elsewhe e (3.2.4.2.1).
R
ESULTS
96
3.5. E ec o Pes icides on he Deg ada ion o Cellulose and
Cellobiose
The pes icides Ben azon, Chlo o halonil, MCPA, Me alaxyl, and Nonylphenol
we e p e-sc eened o hei po en ial o impac on he ae obic and anae obic
deg ada ion o cellobiose (Figu e 24).
Figu e 24. Deg ada ion o supplemen al cellobiose in soil ea men s in he
p esence o pes icides
. (A), (C): Pes icide wi hou appa en e ec (Chlo o halonil,
Me alaxyl). (B), (D): Pes icide wi h appa en e ec (Ben azon, MCPA, Nonylphenol).
Symbols: Ben azon-supplemen ed ea men s; Chlo o halonil-supplemen ed
mic ocosm; MCPA-supplemen ed ea men s; Me alaxyl-supplemen ed ea men s;
Nonylphenol-supplemen ed mic ocosms; Con ol ea men s wi hou pes icides ( ed).
Closed symbols, cellobiose. Open symbols, glucose. E o ba s indica e s anda d de ia ions
(n =3).
Cellobiose was hyd olysed wi hou appa en delay in oxic and anoxic
ea men s, esul ing in an accumula ion o glucose. Pa allel o he hyd olysis o
cellobiose ha las ed 20 hou s in pes icide- ee con ol ea men s, eleased glucose
was consumed. Concen a ions o glucose dec eased in pes icide- ee con ols wi hin
10 hou s (oxic) and 48 hou s (anoxic) o alues unde o nea he de ec ion limi
(~0.5 µmol g
soil DW-1
). Chlo o halonil and Me alaxyl did no appa en ly a ec he
ae obic and anae obic deg ada ion o cellobiose and he subsequen consump ion o
0 6 12 18 24
0.0
0.1
0.2
0.3
0.4
0.0
0.1
0.2
0.3
0.4
A
B
Oxic
0 12 24 36 48
C
D
Anoxic
Time [h]
Cellobiose, Glucose [µmol g
DW-1
]
1.2
0.9
0.6
0.3
0.0
1.2
0.9
0.6
0.3
0.0
0 6 12 18 24
0.0
0.1
0.2
0.3
0.4
0.0
0.1
0.2
0.3
0.4
A
B
Oxic
0 12 24 36 48
C
D
Anoxic
Time [h]
Cellobiose, Glucose [µmol g
DW-1
]
1.2
0.9
0.6
0.3
0.0
1.21.2
0.90.9
0.60.6
0.30.3
0.00.0
1.2
0.9
0.6
0.3
0.0
1.21.2
0.90.9
0.60.6
0.30.3
0.00.0
R
ESULTS
97
glucose (Figu e 24A and C). In con as , he p esence o Ben azon, MCPA, and
Nonylphenol esul ed in a slowe hyd olysis o cellobiose and a s ong inhibi ion o
he subsequen consump ion o glucose (Figu e 24B and D). Comple e cellobiose
hyd olysis was delayed o 4 hou s compa ed o pes icide- ee con ols. Fu he mo e,
he concen a ion o ee glucose doubled in Ben azon-, MCPA-, and Nonylphenol-
supplemen ed ea men s unde oxic and anoxic condi ions esul ing in emaining
glucose a he end o incuba ion (Figu e 24B and D).
Based on hese esul s, Ben azon, MCPA, and Nonylphenol we e chosen as
model pes icides o analyse he impac on he deg ada ion o cellulose and cellobiose
unde oxic and anoxic condi ions (Figu e 25 and Figu e 26; Table 27; Table A2,
2.3.3.3; 2.3.3.2). Di e en concen a ions we e applied o simula e in si u ele an
(‘low’, 3.5.1) and ele a ed (‘high’, 3.5.2) concen a ions o pes icides. Low amoun s o
pes icides we e only used in soil slu ies supplemen ed wi h cellobiose.
3.5.1. In Si u- ele an (‘Low’) Concen a ions o Pes icides
Addi ion o cellobiose s imula ed p oduc ion o ca bon dioxide by app oxima ely
60% and small amoun s o molecula hyd ogen accumula ed unde anoxic condi ions
(Figu e 25). Me hane and e ous i on we e no de ec ed in any o he ea men s. pH
was s able a 6.0 (da a no shown). Supplemen al cellobiose was hyd olysed wi hou
appa en delay in bo h oxic and anoxic ea men s, leading o a ansien
accumula ion o glucose (Figu e 25). Bo h cellobiose and glucose we e o ally
consumed wi hin 10 hou s unde oxic condi ions, and wi hin 42 hou s unde anoxic
condi ions in con ol ea men s lacking pes icides (Figu e 25). Ca bon eco e y was
47% and 55% in oxic and anoxic con ol ea men s, espec i ely. No appa en e ec
on he hyd olysis o cellobiose and he p oduc ion o ca bon dioxide was e iden
unde ‘low’ concen a ion o pes icides (Figu e 25; Table 27; Table A2), whe eas a
sligh inhibi ion o he consump ion o glucose occu ed. Glucose was no o ally
consumed a e 10 hou s in oxic ea men s supplemen ed wi h Ben azon o
Nonylphenol (Figu e 25) wha e e s o an inhibi ion o 12% and 25% by Ben azon
and Nonylphenol, espec i ely (Table 27). Addi ion o pes icides o anoxic ea men s
a ec ed consump ion o glucose mo e p onounced han addi ion o pes icides o oxic
ea men s. Likewise, he o al consump ion o glucose in anoxic ea men s was
slowe in he p esence o pes icides han in pes icide- ee con ol ea men s wha
e e s o an inhibi ion o 24 – 37% (Table 27). P oduc ion o molecula hyd ogen
unde anoxic condi ions was less in he p esence o MCPA and negligible in he
p esence o Ben azon o Nonylphenol a 42 hou s (Table A2).
R
ESULTS
98
Figu e 25. Deg ada ion o supplemen al cellobiose in ea men s unde
‘low‘concen a ion o pes icides.
Pes icide concen a ions: Ben azon: 0.1 µmol g
soil
DW-1
;
Nonylphenol: 0.4 µmol g
soil
DW-1
; MCPA, 0.01 µmol g
soil
DW-1
. Values a e he means o
duplica es. E o ba s indica e s anda d de ia ions. Symbols: ea men s supplemen ed wi h
Ben azon (black), wi h Nonylphenol (whi e), wi h MCPA (g ey), wi hou pes icide
supplemen a ion (g een, dashed line), o wi hou subs a e and addi ion o pes icides ( ed,
do ed line). ca bon dioxide; molecula hyd ogen; ✖ cellobiose; glucose. A ows, ime
poin o calcula ions o inhibi o y e ec o supplemen ed pes icides as p esen ed in Table 27
and Table A2.
3.5.2. Ele a ed (‘High’) Concen a ions o Pes icides
High concen a ions o pes icides simula ed he accumula ion o pes icides ha
can occu in soil by he e ogenous dis ibu ion (Ma sh e al. 1978), especially in
anoxic zones whe e pes icide deg ada ion is slowe compa ed o oxic zones. In
ea men s wi h highe concen a ion o pes icides cellobiose was no comple ely
hyd olysed a e 10 hou s in oxic ea men s (Figu e 26), i.e., a educ ion o he
consump ion o cellobiose o 38%, 38%, and 34% in he p esence o Ben azon,
MCPA, and Nonylphenol, espec i ely, was e iden (Table 27). Likewise, he
consump ion o cellobiose-de i ed glucose and he p oduc ion o ca bon dioxide we e
lowe in oxic ea men s wi h high concen a ion o pes icides compa ed o ea men s
wi h low concen a ion o pes icides and con ols lacking pes icides (Table 27).
Be ween 0.3 ± 0.0 and 0.6 ± 0.0 µmol glucose g
soil DW-1
we e consumed a e 10 days
in oxic pes icide-supplemen ed ea men s whe eas he o al amoun o
1.4 ± 0.1 µmol glucose g
soil DW-1
was consumed in he same pe iod in
CO
2
, H
2
[µmol g
DW-1
]
0
2
4
6
8
Time [h]
0 5 10 15 20 25
Suga s [µmol g
DW
-1
]
0.0
0.4
0.8
1.2
Time [h]
0 10 20 30 40 50
Oxic Anoxic
CO
2
,
H
2
[µmol g
DW-1
]
0
2
4
6
8
Time [h]
0 5 10 15 20 25
Suga s [µmol g
DW
-1
]
0.0
0.4
0.8
1.2
Time [h]
0 10 20 30 40 50
Oxic Anoxic
R
ESULTS
99
unsupplemen ed con ols (Table A3). P oduc ion o ca bon dioxide a high
concen a ion o pes icides was almos as low as he soil indigenous p oduc ion in
subs a e- ee con ol ea men s. In he p esence o Ben azon and MCPA (Figu e 26,
Table A3) a educ ion o 68% and 56% was measu ed unde oxic condi ions (Table
27). Nonylphenol showed only a sligh e ec on he p oduc ion o ca bon dioxide
unde oxic condi ions (Table 27). Supplemen al cellobiose hyd olysis was no
a ec ed in anoxic ea men s supplemen ed wi h high concen a ion o pes icides
(Figu e 26), bu a educed consump ion o glucose was obse ed in he p esence o
Ben azon and MCPA (Figu e 26). A high concen a ion o pes icides, consump ion o
glucose was delayed o mo e han 12 hou s, esul ing in he de ec ion o esidual
glucose a he end o incuba ion (Figu e 26). The amoun o u ilized glucose was 0.2
± 0.1 µmol g
soil DW-1
, and 0.3 ± 0.0 µmol g
soil DW-1
in he Ben azon and MCPA-
supplemen ed ea men s, espec i ely (Table A3), wha e e s o a educ ion o he
consump ion o glucose by mo e han 70% unde high concen a ion o pes icides
(Table 27). In e es ingly, he ini ial anae obic consump ion o glucose was also
slowe unde high Nonylphenol concen a ions, bu accele a ed du ing incuba ion.
This esul ed in simila amoun s o glucose consumed in bo h Nonylphenol-
supplemen ed and unsupplemen ed con ol ea men s (Figu e 26, Table A3).
P oduc ion o ca bon dioxide a high concen a ion o pes icides was educed by
abou 51 – 65% by MCPA and Ben azon, espec i ely (Table 27). No inhibi ion o he
p oduc ion o ca bon dioxide o molecula hyd ogen by Nonylphenol was ob ious
unde anoxic condi ions in which p oduc ion o bo h gaseous compounds was
s imula ed, and concen a ions exceeded con ol ea men s a 42 hou s (Figu e 26).
The anae obic p oduc ion o molecula hyd ogen was negligible in he p esence o
Ben azon and MCPA (Table A3). Fu he mo e, he lack o e men a ion p oduc s in
hese sho incuba ions (Figu e 24 – Figu e 26; 3.5) demons a es ha s ic
anae obes o acul a i e ae obes need ime o o m de ec able amoun s o molecula
hyd ogen, a y acids, o alcohols a e he deple ion o O
2
. A high concen a ion o
pes icides he pH immedia ely dec eased o 5.4 ± 0.6 a e he addi ion o pes icides,
bu s abilized a hese alue o e he incuba ion pe iod (da a no shown).
R
ESULTS
100
CO
2
, H
2
[µmol g
DW-1
]
0
2
4
6
8
10
Time [h]
0 5 10 15 20 25
Suga s [µmol g
DW-1
]
0.0
0.5
1.0
1.5
Time [h]
0 10 20 30 40 50
Oxic Anoxic
CO
2
, H
2
[µmol g
DW-1
]
0
2
4
6
8
10
Time [h]
0 5 10 15 20 25
Suga s [µmol g
DW-1
]
0.0
0.5
1.0
1.5
Time [h]
0 10 20 30 40 50
Oxic Anoxic
Figu e 26. Deg ada ion o supplemen al cellobiose in ea men s unde
‘high‘concen a ion o pes icides
. Pes icide concen a ion: Ben azon: 8.5 µmol g
soil DW-1
;
MCPA, 2.4 µmol g
soil DW-1
; Nonylphenol: 3.5 µmol g
soil DW-1
.
Values a e he means o
duplica es. E o ba s indica e s anda d de ia ions. Symbols: ea men s supplemen ed wi h
Ben azon (black), wi h Nonylphenol (whi e), wi h MCPA (g ey), wi hou pes icide
supplemen a ion (g een, dashed line), o wi hou subs a e and addi ion o pes icides ( ed,
do ed line). ca bon dioxide; molecula hyd ogen; ✖ cellobiose; glucose. A ows, ime
poin o calcula ions o inhibi o y e ec o supplemen ed pes icides as p esen ed in Table 27
and Table A3.
The impac o pes icides on he deg ada ion cellulose was in es iga ed by
inse ion o cellulose pape shee s in o sie ed esh soil and des uc i e sampling,
i.e., emo ing o one eplica e om he analysis, e e y 7 days. App oxima ely 50% o
he supplemen ed cellulose was deg aded du ing he 70-day incuba ion unde bo h
oxic and anoxic condi ions in pes icide- ee con ol ea men s, i.e., abou 12 µmol
cellulose g
soil DW-1
was consumed (Figu e 27, Table A3). Hyd oly ic p oduc s o
cellulose (cellobiose o glucose) we e no de ec ed in ei he oxic o anoxic ea men s
(de ec ion limi s app oxima ed 0.5 µmol g
soilDW-1
). O ganic p oduc s we e no de ec ed
du ing he i s ou weeks, bu small amoun s o ace a e, p opiona e, and bu y a e
accumula ed in anoxic cellulose-supplemen ed ea men s (< 5 µmol g
soil DW-1
) lacking
pes icides, bu no in oxic ea men s. P oduc ion o ca bon dioxide was s imula ed up
o 50% by he addi ion o cellulose unde bo h oxic and anoxic condi ions wi hou
pes icides (Figu e 27, Table A3). Ca bon eco e y was 106% and 81% in oxic and
anoxic con ol ea men s, espec i ely. Addi ion o cellulose also s imula ed e ic
i on educ ion unde anoxic condi ions. Fe ous i on eached concen a ions o up o
abou 190 µmol g
soil DW-1
. In ea men s wi hou subs a e, e ous i on was no
R
ESULTS
101
de ec ed (Figu e 27, Table A3). Molecula hyd ogen and me hane we e no de ec ed
in any o he cellulose-supplemen ed ea men s. pH was s able a 6.2 (da a no
shown).
Ben azon, MCPA, and Nonylphenol a 2.4 µmol g
soil DW-1
impai ed he
deg ada ion o cellulose unde oxic condi ions by 41%, 62%, and 47%, espec i ely.
Ben azon and MCPA educed p oduc ion o ca bon dioxide by 20 – 32% (Table 27).
Nonylphenol had no appa en inhibi o y e ec (Figu e 27). In con as , Nonylphenol
s imula ed p oduc ion o ca bon dioxide unde oxic condi ions (Table A3) wha can be
explained by he deg ada ion o his pes icide. A e 28 weeks 2.4 µmol
Nonylphenol g
soil DW-1
we e o ally consumed in oxic ea men s making a second
applica ion o Nonylphenol o emaining eplica es nesseca y (Figu e 27).
Concen a ions o Ben azon and MCPA dec eased slowly in oxic cellulose-
supplemen ed ea men s, esul ing in abou 50% esidual pes icide a he end o
incuba ion (Figu e 27).
Pes icide-impai ed deg ada ion o supplemen al cellulose was mo e e iden
unde anoxic han unde oxic condi ions, i.e. less han 1 µmol cellulose g
soil
DW-1
was
consumed (Figu e 27, Table A3). This co esponds o an inhibi ion o mo e han 90%
by all pes icides (Table 27). Accumula ion o o ganic p oduc s (i.e., ace a e,
p opiona e, and bu y a e) did no exceed 1.5 µmol g
soil
DW-1
(da a no shown) and
p oduc ion o ca bon dioxide was impai ed by 85%, 85%, and 44% o Ben azon,
MCPA, and Nonylphenol, espec i ely (Table 27). Reduc ion o e ic i on was also
educed, i.e., 60 – 96% less e ous i on accumula ed in he p esence o pes icides
(Table A3). Concen a ion o pes icides was s able in anoxic cellulose-supplemen ed
ea men s (Figu e 27).
Figu e 27. Deg ada ion o supplemen al cellulose in soil ea men s in he
p esence o pes icides a 2.4 µmol g
soil DW-1
.
T
end
alues a e he means o iplica es.
E o ba s indica e s anda d de ia ions. Symbols: ea men s supplemen ed wi h Ben azon
CO
2
[µmol g
DW-1
]
0
50
100
150
Pes icide [µmol g
DW-1
]
0
1
2
3
Time [d]
0 14 28 42 56 70
Cellulose [µmol g
DW-1
]
0
10
20
30
Time [d]
0 14 28 42 56 70
Fe
2+
[µmol g
DW-1
]
0
50
100
150
Oxic Anoxic
CO
2
[µmol g
DW-1
]
0
50
100
150
Pes icide [µmol g
DW-1
]
0
1
2
3
Time [d]
0 14 28 42 56 70
Cellulose [µmol g
DW-1
]
0
10
20
30
Time [d]
0 14 28 42 56 70
Fe
2+
[µmol g
DW-1
]
0
50
100
150
Oxic Anoxic
R
ESULTS
102
(black), wi h MCPA (g ey), wi h Nonylphenol (whi e), and wi hou pes icide supplemen a ion
(g een, dashed line), o wi hou subs a e and addi ion o pes icides ( ed, do ed line);
ca bon dioxide; ▼ pes icide (dashed line); cellulose; ▲ e ous i on. A ows, ime poin o
calcula ions o inhibi o y e ec o supplemen ed pes icides as p esen ed in Table 27 and
Table A3. Nonylphenol was applied wice (day 0, day 28) in oxic ea men s.
Table 27. Inhibi ion o saccha ide deg ada ion, ca bon dioxide and hyd ogen
emission, and p oduc ion o e ous i on in he p esence o pes icides in soil
ea men s compa ed o con ol ea men s.
Inhibi ion [%]
Oxic
Anoxic
Ben azon
MCPA
Nonylphenol
Ben azon
MCPA
Nonylphenol
‘
Low’ concen a ion
o
pes icides
–
Cellobiose
-
supplemen ed
a
Cellobiose
consump ion
d
11.1 ±
9.5 n.i. 15.4 ±
6.3
n.i. n.i. n.i.
Glucose
consump ion
d
12.2 ±
6.3 n.i. 25.1 ±
8.4
26.6 ±
13.5 23.5 ±
2.5 37.3 ±
2.3
CO
2
p oduc ion
d
n.i. n.i. n.i.
13.6 ±
11.3 13.4 ±
1.4 n.i.
H
2
p oduc ion
d
- - -
100.0 ±
0.0 89.2 ±
1.5 100.0 ±
0.0
‘
High’
concen a ion o
pes ici
des
–
Cellobiose
-
supplemen ed
b
Cellobiose
consump ion
d
38.3 ±
7.0 37.8 ±
6.0 34.4 ±
0.3
n.i. n.i. n.i.
Glucose
consump ion
d
81.0 ±
0.1 59.1 ±
4.3 61.1 ±
7.5
81.8 ±
6.0 73.4 ±
0.9 n.i.
CO
2
p oduc ion
d
68.2 ±
3.3 55.6 ±
5.3 32.6 ±
2.4
64.7 ±
5.1 51.0 ±
5.9 n.i.
H
2
p oduc ion
d
- - -
100.0 ±
0.0 100.0
± 0.0 n.i.
‘High’
concen a ion o
pes icides
–
Cellulose
-
supplemen ed
c
Cellulose
deg ada ion
e
40.8 ±
12.0 61.6 ±
2.6 47.4 ±
11.0
94.8 ±
5.0 93.8 ±
3.6 97.0 ±
3.4
CO
2
p oduc ion
e
20.3 ±
6.5 31.5 ±
4.5 n.i.
84.6 ±
0.8 85.6 ±
2.1 44.2 ±
12.8
Fe
2+
p oduc ion
e
- - -
95.7 ±
0.5 95.4 ±
0.3 59.9 ±
1.0
R
ESULTS
103
a
0.01 o 0.4 µmol pes icide g
soil
DW-1
.
b
2.4 o 8.5 µmol pes icide g
soil
DW-1
.
c
2.4 µmol pes icide g
soil
DW-1
.
d
Inhibi ion o saccha ide deg ada ion, ca bon dioxide and hyd ogen emission was calcula ed
based on he di e ences in he concen a ion o p oduc s a 10 hou s (oxic) and 42 hou s
(anoxic) e e ed o he con ol ea men (Table A2).
e
Inhibi ion o cellulose deg ada ion, ca bon dioxide and hyd ogen emission, and e ic i on
educ ion was calcula ed based on he di e ences in he concen a ion o p oduc s a 70
days o incuba ion e e ed o he con ol ea men (Table A3).
.i., No inhibi ion, i.e., amoun o p oduc was highe in pes icide-supplemen ed ea men s
han in con ol ea men s o s anda d de ia ions whe e highe han he mean alue.
-, No de e mined.
3.5.3. 16S RNA Con en o Bac e ial Taxa
16S RNA ansc ip numbe s o o al soil Bac e ia and i e saccha ide-u ilizing
amily-le el axa (Mic ococcaceae/Cellulomonadaceae [Ac inobac e ia], Clus e I
Clos idiaceae [Fi micu es], Planc omyce aceae [Planc omyce es], and he new axa
‘Cellu1-3’ and ‘Sphingo1-4’ [Bac e oide es]), we e de e mined in soil samples o
cellulose-supplemen ed ea men s using e e se ansc ip ase quan i a i e PCR
(RTqPCR). Taxa we e quan i ied in Ben azon-supplemen ed, MCPA-supplemen ed,
and pes icide- ee con ol ea men s, bu no in Nonylphenol-supplemen ed
ea men s.
RNA con en o he o al bac e ial soil communi y dec eased in all ea men s
om 6.4 × 10
10
ansc ip s ng
RNA-1
o signi ican ly lowe alues (Figu e 28)
independen o he p esence o absence o pes icides. This consis en dec ease
migh be explained by a ious ac o s, e.g., he dea h o non-saccha ide-u ilizing
bac e ia due o subs a e limi a ion o g azing o Bac e ia by euka yo es eeding on
bac e ia (e.g., p o ozoa, cilia es). Ben azon and MCPA applica ion lowe ed he
numbe o ansc ip o o al soil Bac e ia in bo h oxic and anoxic ea men s a he
end o he expe imen compa ed o pes icide- ee ea men s (Figu e 28). RNA
con en dec eased om 3.8 × 10
10
ansc ip s ng
RNA-1
in pes icide- ee con ols o
1.5 × 10
10
ansc ip s ng
RNA-1
in Ben azon-supplemen ed, and 1.2 × 10
10
ansc ip s
ng
RNA-1
in MCPA-supplemen ed ea men s (Fig. 31) unde oxic condi ions. In anoxic
ea men s Ben azon and MCPA applica ion lowe ed he numbe o 16S RNA genes
om 3.0 × 10
10
ansc ip s ng
RNA-1
o 1.3 × 10
10
ansc ip s ng
RNA-1
and
0.6 × 10
10
ansc ip s ng
RNA-1
, espec i ely (Figu e 28).
R
ESULTS
110
T ansc ip s ng
RNA-1
048 102 168 192
Time [h]
10
10
10
8
10
6
10
4
*
**
*
*
*
*
Figu e 31. Numbe s o 16S RNA gene ansc ip s o Bac e ia and A chaea in
ea men s in incuba ion chambe s
. The illed ba s ep esen alues ob ained om
CMC-supplemen ed ea men s. The shaded ba s ep esen alues ob ained om he
cellobiose-supplemen ed ea men s. The emp y ba s ep esen alues ob ained om
unsupplemen ed con ol ea men s. E o ba s indica e s anda d de ia ions (n = 3). Ba
code: non-ha ched, Bac e ia; ha ched, A chaea. An as e isk indica es ha a ansc ip
numbe is s a is ically di e en om he ansc ip numbe o he p e ious ime poin ( -Tes ).
The syn hesis o 16S RNA o amily-le el axa was di e en ially a ec ed by
addi ion o CMC and cellobiose (Figu e 32 and Table A1). A selec i e ac i a ion
and/o ep ession o unc ionally edundan axa was obse ed (Figu e 32). No
amily-le el axon was s imula ed wi hou addi ion o subs a e, i.e., he amoun o
16S RNA genes did no change signi ican ly in unsupplemen ed con ols (Table A1).
The ela i e amoun o 16S RNA o Mic ococcaceae/Cellulomonadaceae inc eased
signi ican ly om 9.8 × 10
-5
o 5.6 × 10
-5
ansc ip s ansc ip s
Bac e ia-1
in he i s oxic
pe iod o cellobiose-supplemen ed ea men s, bu no in CMC-supplemen ed
ea men s (Figu e 32A, Table A1). Anoxic incuba ion (48 – 168 hou s) esul ed in a
signi ican dec ease o ansc ip numbe s o Mic ococcaceae/Cellulomonadaceae in
cellobiose-supplemen ed ea men s (Figu e 32A). 16S RNA gene ansc ip
numbe s o Planc omyce aceae beha ed in e sely (Figu e 32D). Addi ion o
cellobiose did appa en ly no a ec RNA con en o Planc omyce aceae (Figu e 32D,
Table 24), bu addi ion o CMC esul ed in a signi ican inc ease om 1.4 × 10
-6
o 1.8
× 10
-5
ansc ip s ansc ip s
Bac e ia-1
du ing e-ae a ion (Table A1). Membe s o
Kineospo iaceae/Noca dioidaceae we e no s imula ed by ei he CMC o cellobiose
(Figu e 32B). In con as , ela i e ansc ip numbe s o his axon dec eased du ing
he oxic and he i s hal o he anoxic pe iod (0 – 168 hou s) om 6.3 × 10
-7
o
2.1 × 10
-7
ansc ip s ansc ip s
Bac e ia-1
and om 9.3 × 10
-7
o 1.3 × 10
-7
ansc ip s
ansc ip s
Bac e ia-1
in CMC- and cellobiose-supplemen ed ea men s, espec i ely
(Table A1). Simila esul s we e ob ained o he uncul u ed axon ‘Deha1’ (phylum
Chlo o lexi), o which s imula ion by addi ion o subs a es was no appa en (Figu e
32F, Table A1). T ansc ip numbe s o Clus e I Clos idiaceae and he new amily-
le el axon ‘Cellu1-3’ (phylum Bac e oide es) inc eased unde anoxic condi ions
R
ESULTS
111
(48 - 68 h) in cellobiose-supplemen ed ea men s bu no unde oxic condi ions
(Figu e 32C and E). T ansc ip numbe s o Clus e I Clos idiaceae and ‘Cellu1-3’
inc eased o 1.9 × 10
-7
and 1.0 × 10
-7
ansc ip s ansc ip s
Bac e ia-1
a e 168 hou s o
incuba ion in he p esence o cellobiose, espec i ely (Table A1). CMC did no
appea o s imula e hese axa, nei he in he oxic pe iod no in he anoxic pe iod
(Figu e 32C and E, Table A1). The uncul u ed axon ‘Sphingo1-4’ (phylum
Bac e oide es) was he only axon ha was sligh ly s imula ed by bo h subs a es
(Figu e 32G, Table A1). In he second hal o he anoxic pe iod 16S RNA genes
inc eased om 8.4 × 10
-9
o 2.2 × 10
-8
ansc ip s ansc ip s
Bac e ia-1
and om
2.1 × 10
-6
o 4.5 × 10
-6
ansc ip s ansc ip s
Bac e ia-1
in CMC- and cellobiose-
supplemen ed ea men s, espec i ely (Table A1).
R
ESULTS
112
Figu e 32. E ec s o CMC and cellobiose on axon-speci ic 16S RNA gene
ansc ip s.
The shaded a eas co espond o he anoxic pe iods. Symbols: illed, alues
ob ained om CMC-supplemen ed ea men s; g ey, alues ob ained om cellobiose-
supplemen ed ea men s; emp y, alues ob ained om unsupplemen ed con ol ea men s.
Values a e he means o wo eplica es (wi hou s anda d de ia ion) o h ee eplica es (wi h
s anda d de ia ion). The highes numbe o axon-speci ic ansc ip s pe assay (i.e., in CMC,
cellobiose, and unsupplemen ed con ol ea men s) was se a 100% (absolu e and e e ence
alues a e ma ked in Table A1). An as e isk indica es ha a ansc ip numbe is s a is ically
di e en om he ansc ip numbe o he p e ious ime poin ( -Tes ).
0
50
100
150
0
50
100
150
0 50 100 150 200
0
50
100
150
A Mic ococca-
/Cellulomonadaceae B Kineospo ia-
/Noca dioidaceae
C Clus e I
Clos idiaceae D Planc omyce aceae
E ‘Cellu1-3’ F ‘Deha1’
0 50 100 150 200
0
1
50
100
150
G ‘Sphingo1-4’
*
**
*
*
*
*
*
*
*
*
*
**
Change in T ansc ip s [%]
**
*
Time [h]
Time [h]
R
ESULTS
113
A shi in he a io o amily-le el axa was also obse ed.
Mic ococcaceae/Cellulomonadaceae became he mos abundan o all de ec ed
g oups in cellobiose-supplemen ed ea men s a e he ini ial oxic phase. Such an
en ichmen o RNA o hese Ac inobac e ia was no obse ed in CMC-supplemen ed
ea men s (Figu e 33). In con as , 16S RNA cDNA genes o he new amily-le el
axon ‘Deha1’ (Chlo o lexi) comp ised a high p opo ion o he au och honous soil
mic obial communi y (Figu e 33), bu nei he his axon no o he de ec ed axa we e
ema kably ac i a ed in CMC- o cellobiose-supplemen ed ea men s (Figu e 32).
Figu e 33. Rela i e abundances o amily-le el axa in (A) CMC-
supplemen ed ea men s, (B) cellobiose-supplemen ed ea men s, and (C)
unsupplemen ed con ol ea men s.
100% co esponds o he o al numbe o
ansc ip s o all axa (numbe s abo e ba s in [× 10
-3
ansc ip s ng
RNA-1
]).
Time [h]
0 48 102 168 192
[%]
0
20
40
60
80
100
0.9 0.40.5 0.5
0.7
A
Time [h]
0 48 102 168 192
1.4 6.8 6.2 3.9 4.4
B
Time [h]
0 48 102 168 192
[%]
0
20
40
60
80
100
1.2 0.9 0.8 0.9 0.7
C
Clos idia III
'Cellu' (
‘Cellu1-3‘ (Bac e oide es)
Clus e I Clos idiaceae
‘Deha1‘ (Chlo o lexi)
Kineospo iaceae & Noca dioidaceae
Kineospo ia-/Noca dioidaceae
Mic ococcaceae & Cellulomonadaceae
Clos idia
Mic ococca-/Cellulomonadaceae
Planc omyce aceae
‘Sphingo1-4‘ (Bac e oide es)
%
%
D
ISCUSSION
114
4.
D
ISCUSSION
Cellulose is he mos abundan biopolyme on ea h and i s biological
deg ada ion in soil is a majo p ocess in he global ca bon cycle (Baye e al. 2006;
Falkowski e al. 2000; Lal 2008; Lynd e al. 2002). Ag icul u al soils accoun o abou
40% o he wo ld’s e es ial su ace a ea and a e ho spo s o he u no e o
o ganic ca bon (Kö be e al. 2009). Ag icul u al soil is a highly s uc u ed
en i onmen in which oxic and anoxic mic ozones co-occu on a small scale (Figu e
3), and in which he dis ibu ion and dimension o such zones can change apidly
(Dasson ille e al. 2004; O e al. 2007; Six e al. 2000; To sche e al. 2010; Zausig e
al. 1993). This compa men aliza ion acili a es he coexis ence and simul aneous
ac i i y o ae obic and anae obic soil mic oo ganisms ha ca alyse he deg ada ion o
plan -de i ed cellulose unde con as ing a ailabili ies o O
2
. Howe e , less is known
abou he phylogene ic iden i ies o cellulose-deg ading mic obial communi ies in si u
and hei me abolic esponse o apid changes in O
2
. Ano he c ucial ac o is he
inc easing usage o pes icides in ag icul u al ecosys ems. Applica ion o he bicides,
ungicides, and insec icides has esul ed in an accumula ion o pes icide esidues in
e es ial and aqua ic habi a s (Ake blom 2004; Hille e al. 2008; Tho s ensen e al.
2001). Pes icides can impac on soil communi ies (Cox e al. 1996; Johnsen e al.
2001; Ka ayama and Kuwa suka 1991; Po e and Hayden 2002), and migh al e he
cellulose-dependen ca bon low and he ac i i y o in ol ed mic obial axa in
ag icul u al ecosys ems. T ophic in e ac ions be ween cellulose-deg ading
mic oo ganisms a e complex and many uncul u ed species a e likely impo an o he
deg ada ion o plan -de i ed ca bon (Haicha e al. 2007; Li e al. 2009). To
unde s and he p ocess o cellulose deg ada ion in such a dynamic en i onmen , i is
essen ial (i) o iden i y in ol ed cellulose-deg ading soil communi ies and (ii) o
in es iga e he impac o O
2
and pes icides on ac i e mic obial axa.
4.1. Ae obic and Anae obic Deg ada ion o Saccha ides in An
Ae a ed Ag icul u al Soil
A communi y unc ion, e.g., he u iliza ion o glucose o he u no e o ni ogen,
may emain s able unde changing O
2
s a es (Picek e al. 2000; San ucko a e al.
2004) al hough he composi ion o he mic obial communi y shi s (Pe -Ridge and
Fi es one 2005; Pe -Ridge e al. 2006). In his ega d, he capaci y o he saccha ide-
deg ading communi y o eadily consume supplemen al subs a es was no a ec ed
by he a ailabili y o O
2
o changing edox po en ials (Figu e 11, Figu e 27, Figu e
30). Supplemen ed ca boxyme hyl-cellulose (CMC) was consumed wi hou appa en
delay in oxic and anoxic ea men s (Figu e 30A), indica ing ha he saccha ide-
deg ading communi y o he soil was poised o bo h ae obically and anae obically
consume his subs a e. In con as o CMC-supplemen ed ea men s, a sho lag
phase was obse ed in ea men s ha ecei ed cellulose in he insoluble o m, i.e.,
cellulosic pape shee s (Figu e 27). The lag phase be o e measu able deg ada ion o
cellulose pape sugges s an ini ial coloniza ion o he subs a e by celluloly ic
mic oo ganisms p io o i s biological deg ada ion (Munie -Lamy and Bo de 2000). A
lowe abundance o accessible egions o enzyma ic hyd olysis in c ys alline
cellulose compa ed o soluble cellulose-de i a es migh also accoun o he slow
deg ada ion o cellulose pape . Endo- and exoglucanases exc e ed by celluloly ic
mic oo ganisms a ack c ys alline cellulose a in e nal amo phous egions, and
inc ease he amoun s o educing and non- educing chain ends (Baye e al. 1998a;
Baye e al. 1998b; Bisa ia and Ghose 1981; Lynd e al. 2002; Tee i 1997; Figu e 2).
D
ISCUSSION
115
Thus, p olonged incuba ion p omo es g ow h o celluloly ic mic oo ganisms on
cellulose shee s by an inc ease o subs a e accessibili y. The lack o de ec able
cellodex ins, cellobiose, and glucose du ing cellulose deg ada ion (Figu e 11A,
Figu e 27, Figu e 30A) sugges s a e y e icien assimila ion o eleased hyd olysis
p oduc s by celluloly ic mic oo ganisms and saccha oly ic sa elli e o ganisms (Baye
e al. 1994; Baye e al. 2004; S anie 1942; Xie e al. 2007; Figu e 4, 1.5), especially
unde oxic condi ions. Unde anoxic condi ions, anae obic celluloly ic mic oo ganisms
a ack cellulosic subs a es mainly by an ex acellula enzyme complex (i.e.,
cellulosome; 1.2.1.2) ha is co alen ly linked wi h he cell en elope and a aches
igh ly o he subs a e by ca bohyd a e binding domains (Baye e al. 1998b; Beguin
and Aube 1994; Des aux 2005b; Lynd e al. 2002). Thus, he elease o soluble
hyd olysis p oduc s is minimal (Baye e al. 1994; Beguin and Aube 1994).
Cellobiose was hyd olyzed, esul ing in a ansien accumula ion o glucose
(Figu e 11B, Figu e 24 – Figu e 26, Figu e 30B). This phenomenon may ha e been
caused by ex acellula β-glucosidases ha hyd olyzed cellobiose as e han glucose
was consumed by mic oo ganisms (Hong e al. 1981; Lynd e al. 2002). Cellobiose
also accumula ed in oxic s able iso ope p obing ea men s wi h p olonged incuba ion
(Figu e 11B) wha sugges s a subsequen p oduc -media ed inhibi ion o cellobiose
hyd olysis/up ake by inc easing concen a ions o glucose (Co azza e al. 2005;
Gong e al. 1977; Kajikawa and Masaki 1999). Howe e , i emains unclea , why he
hyd olysis o cellobiose was inhibi ed in oxic ea men s o s able iso ope p obing
expe imen , bu no in o he expe imen s. Ne e heless, supplemen ed cellobiose
and glucose we e ae obically and anae obically consumed. Ca bon eco e y was
below 60% in oxic ea men s supplemen ed wi h cellobiose o glucose (3.1, 3.5)
which indica es ha a subs an ial amoun o ca bon was assimila ed du ing ae obic
con e sion. This end is consis en wi h s udies ha assessed he inco po a ion o
ca bon om [
13
C]- and [
14
C]-labeled cellulose, cellobiose, o glucose in o mic obial
biomass o o es , sa annah, and loamy soil (DeFo es e al. 2004; Fon aine e al.
2004; Schneckenbe ge e al. 2008). Acco ding o li e a u e, ca bon dioxide was he
main end p oduc o he deg ada ion o supplemen ed saccha ides unde oxic
condi ions (Baye e al. 2006; Beguin 1990, Beguin and Aube 1994; Schmid and
Ruschmeye 1958). These esul s demons a e ha p ocesses ha a e linked o he
ae obic deg ada ion o saccha ides a e simila in a ious ypes o soil and migh be
ca alyzed by mic obial communi ies wi h simila ecosys em unc ions.
Ca bon eco e y was abo e 100% in anoxic ea men s o s able iso ope
p obing expe imen s (3.1) sugges ing ha supplemen al subs a es augmen ed he
concomi an u iliza ion o soil indigenous ca bon and enhanced u no e o mic obial
biomass (Blagoda skaya and Kuzyako 2008; de Nobili e al. 2001). This so-called
‘P iming E ec ’ esul s in inc eased p oduc ion o ca bon dioxide
due o s imula ion o
deg ada ion o al eady a ailable ca bon compounds by soil mic oo ganisms a e
addi ion o [
13
C]-cellulose o o he o ganic ca bon compounds (Blagoda skaya e al.
2007; Blagoda skaya and Kuzyako 2008; Fon aine e al. 2004; Zyakun and Dilly
2005). P oduc s ha a e indica i e o e men a i e me abolisms accumula ed unde
anoxic condi ions (Figu e 11, Figu e 30), a esul consis en wi h p e ious labo a o y
s udies ha ha e in es iga ed he e ec s o O
2
limi a ion on ae a ed soils
(Degelmann e al. 2009a; Küsel and D ake 1995). In looded o we land soils,
cellulose, and i s ini ial main b eakdown p oduc s cellobiose and glucose a e
decomposed by anae obes in o a y acids, alcohols, molecula hyd ogen, and
ca bon dioxide, physiological e en s ha can be coupled o ace ogenesis and
me hanogenesis (D ake e al. 2009; Wes e mann 1996). Howe e , hyd ogeno ophic
D
ISCUSSION
116
ace ogenesis (i.e., he hyd ogen-dependen educ ion o ca bon dioxide o ace a e by
ace ogens [D ake e al. 2006; D ake e al. 2008]) was likely o mino impo ance
since he headspace was egula ly lushed wi h dini ogen (2.3.1). The p oduc ion o
ace a e, bu y a e, p opiona e, and molecula hyd ogen (Figu e 11, Figu e 30, 3.1,
3.6.1) sugges s ha a combina ion o e men a ion ypes was ac i e unde anoxic
condi ions, including mixed acid e men a ion and Clos idia- ypical p opiona e o
bu y a e e men a ion (Buckel 2005; Go schalk 1986; Whi e 2007). These
e men a i e me abolisms a e likely sou ces o molecula hyd ogen (Buckel 2005;
Whi e 2007). In con as o o es soils ha can o m huge amoun s o succina e and
e hanol ia En e obac e iaceae- acili a ed mixed acid e men a ion unde anoxic
condi ions (Degelmann e al. 2009a), he ag icul u al soil in es iga ed in he p esen
s udy did no yield hese p oduc s in signi ican amoun s, sugges ing ha hey we e
no main p oduc s o suga e men a ion unde expe imen al condi ions o subjec ed
o consump ion. This co esponds o he low ela i e abundance o ac i e
En e obac e iaceae in anoxic ea men s (Table 22). In con as , addi ion o o ganic
ca bon o anoxic soil (i.e., ice ield o en soil) esul s in an immedia e accumula ion
o molecula hyd ogen, ace a e, and p opiona e (Hambe ge e al. 2008; Penning and
Con ad 2007; Wüs e al. 2009). In e es ingly, supplemen a ion o cellobiose
s imula ed he p oduc ion o lac a e unde anoxic condi ions in ea men s ha we e
subjec ed o luc ua ing a ailabili ies o O
2
(2.3.2), and lac a e was no apidly
consumed a e e-ae a ion (Figu e 30B). In con as , lac a e was no a de ec able
compound in s able iso ope p obing expe imen s (Figu e 11; 3.1). Ins ead, p opiona e
accumula ed (Figu e 11). P opiona e is classically p oduced by s ic anae obic
bac e ia o he P opionibac e iaceae (Ac inobac e ia), Veillonellaceae, Clos idiaceae,
and ‘S ep opep ococcaeae’ (Fi micu es; G aham e al. 2011). P opiona e-p oducing
bac e ia can e men glucose, alcohols, and some amino acids o p opiona e
(Pi e eau 1999), bu hei p e e ed subs a e is lac a e (G aham e al. 2011). Lac a e
can ei he be educed ia he ac yla e pa hway o ace a e and p opiona e, o
con e ed s epwise ia he succina e-p opiona e pa hway o succina e and
p opiona e (G aham e al. 2011). Succina e can be an in e media e o an end p oduc
o P opionibac e ia- ela ed e men a ion (Pi e eau 1999). Nei he succina e no
P opionibac e ia- ela ed sequences we e de ec ed in s able iso ope p obing
expe imen s (Figu e 11, Table 19, Table 20, Table 23) wha sugges s ha he
p oduc ion o p opiona e was no ca alyzed by P opionibac e iaceae. The lack o
lac a e and succina e du ing long anoxic incuba ion (up o 84 days in s able iso ope
p obing expe imen s; 3.1) and hei de ec ion du ing sho e anoxic incuba ion
(< 10 days; 3.6.1) indica es ha hese compounds a e in e media es o he
deg ada ion o saccha ides, a e apidly u ned o e , and likely unc ion as p ecu so s
o p opiona e- and ace a e- o ma ion by he mic obial soil communi y. Fo ma e ha
was p oduced in aces du ing anoxic pe iods was also no a s able in e media e
(da a no shown). A simila ou e o ca bon u no e is also supposed o be ele an
in en soil o in he gu o ea hwo ms (Hambe ge e al. 2008; Wüs e al. 2011).
Pa allel o he deg ada ion o supplemen ed saccha ides ni a e was consumed
in mos oxic and anoxic incuba ions (Figu e 29, Figu e 30; 3.1, 3.6.1). This indica es
ha ni a e was subjec ed o assimila ion and dissimila ion. Ae obic and anae obic
assimila ion o ni a e ia ni i e (NO
2-
) and ammonium (NH
4+
) in o biomass (Fuchs
2007; Rudol and K oneck 2005; Tiedje 1988), anae obic dissimila ion o ni a e
o
ni i e (DNR; S olz and Basu 2002) o dini ogen (deni i ica ion; Payne 1981), and
anae obic dissimila ion o ni a e o ammonium (DNRA; Fuchs 2007; Simon 2002)
may accoun o he consump ion o ni a e. The capabili y o assimila e ni a e is
common o mos bac e ia, ungi, algae, and highe plan s, and necessa y o
D
ISCUSSION
117
inco po a e ni ogen in o cellula componen s (Rudol and K oneck 2005). In con as ,
dissimila ion o ni a e is ca alyzed almos exclusi ely by P oka yo es and is
pe o med o gain ene gy unde anoxic condi ions (Tiedje 1988). Concen a ions o
ni a e inc eased in CMC-supplemen ed and unsupplemen ed ea men s du ing he
oxic pe iod (3.6.1), and dec eased unde anoxic condi ions (Figu e 29, Figu e 30).
This sugges s ha he p oduc ion o ni a e by ni i ica ion exceeded he consump ion
o ni a e (Sp en e al. 1987; Yu e al. 2007), and ha ni a e was anae obically
assimila ed o u ilized as elec on sink by he soil communi y ia dissimila o y
pa hways (Con ad 1996, Dasson ille e al. 2004; Pe e s and Con ad 1996; Yu e al.
2007). A ailable ni a e migh also accoun o he appa en lack o e ous i on and
e men a ion p oduc o ma ion (Dasson ille e al. 2004; Picek e al. 2000).
The modynamically high edox po en ials do no a o mos e men a ions and he
educ ion o ni a e o e ic i on (Bohn 1971). The educ ion o ni a e and e ic i on
in wa e -unsa u a ed bulk soil a he occu s a 230 mV and 150 mV, espec i ely
(Mans eld 2004). Values o 300 mV (ni a e educ ion) and 50 mV ( e ic i on
educ ion) a e measu ed in wa e -sa u a ed ice ield soil, espec i ely (Yu e al.
2007). Mos e men a ions (excep lac a e e men a ion by ae o ole an lac ic acid
bac e ia; B ioukhano and Ne uso 2007; Chen e al. 2005; Ma hies e al. 2004)
occu op imally a edox po en ials below -100 mV (Dasson ille e al. 2004; Picek e
al. 2000). Hence, he p esence o ni a e in CMC-supplemen ed and unsupplemen ed
ea men s migh ha e been esponsible o he s abiliza ion o edox po en ials
abo e 320 mV (Figu e 29, Figu e 30A).
In con as o CMC- and unsupplemen ed ea men s, a apid dec ease o he
edox po en ial in cellobiose-supplemen ed ea men s occu ed when O
2
was
emo ed. The edox po en ial d opped mo e han 700 mV wi hin 10 h (Figu e 30B). I
is likely ha his change in edox po en ial was due o he me abolic ac i i y o
saccha ide-u ilizing mic obes ha coupled he oxida ion o saccha ides o he
educ ion o high edox po en ial elec on accep o s such as oxygen, ni a e, and
e ic i on (Figu e 11, Figu e 30B; Dasson ille e al. 2004; Küsel e al. 2002; Lo ley e
al. 2004; Pe e s and Con ad 1996). This conclusion is consis en wi h he labeling o
s ic anae obic and acul a i e ae obic axa capable o he educ ion o ni a e o
e ic i on. Fo example, species o Cellulomonadaceae, Rhodocyclaceae,
Clos idiaceae, Geobac e aceae, Pelobac e aceae and Ae omonadaceae pe o m
deni i ica ion, educe ni a e o ni i e o ammonium, o dissimila e e ic i on unde
anoxic condi ions (Colwell e al. 1986; Kang e al. 2007; Lee 2006a; Lo ley e al.
2004; Shida e al. 1997; Tiedje 1988; an Keulen e al. 2007; Wiegel e al. 2006). I is
gene ally supposed ha mic oo ganisms ha educe e ic i on do no di ec ly u ilize
complex o ganic ma e (Lo ley 2006; Lo ley e al. 2004), bu p e e ibly u ilize
e men a ion p oduc s, such as a y acids and alcohols, as elec on dono s (Lo ley e
al. 1993; Lo ley e al. 1995). Howe e , some i on educe s can also u ilize glucose
(e.g., Ae omonadaceae, Clos idiaceae; Dobbin e al. 1999; Pham e al. 2003).
Hence, i can be assumed ha species o Ae omonadaceae and Clos idiaceae
ca alyzed he saccha oly ic educ ion o e ic i on in he expe imen s.
Concomi an o he educ ion o al e na i e elec on accep o s wi h posi i e
edox po en ials and he high amoun s o eadily accessible o ganic ca bon, he
dec easing edox po en ial a o ed e men a ion p ocesses in cellobiose-
supplemen ed ea men s du ing he anoxic pe iod (Figu e 30B). Re-ae a ion o
cellobiose-supplemen ed ea men s esul ed in a s ong inc ease o he edox
po en ial and as consump ion o a y acids and e ic i on (Figu e 30), he la e
p ocesses likely being media ed by he e o ophic ae obes.
D
ISCUSSION
118
Me hane was no p oduced in any expe imen unde anoxic condi ions. This
esul is in con as o we land soils ha yield me hane om saccha ides ia
‘in e media y ecosys em me abolism’ (i.e., by he ophically linked p ocesses ha
p ecede me hanogenesis [D ake e al. 2009]; 1.2.2). The p oduc ion o me hane in
acidic en soil can be s imula ed by me hanogenic p ecu so s like o ma e o ca bon
dioxide, and non-me hanogenic p ecu so s like xylose o glucose (Hambe ge e al.
2008; Hunge e al. 2011). This is coinciden wi h he occu ence o ac i e
me hanogens and ypical e men ing species in en soil, sugges ing ha e men a ion
p ocesses and me hanogensis a e ophically linked (Hambe ge e al. 2008; Hunge
e al. 2011). Fu he mo e, me hanogens can be cul i a ed om ae a ed soils (Pe e s
and Con ad 1995), and some species ole a e oxygen (K ä ze e al. 2011; Liu e al.
2008; Tholen e al. 2007). Me hane p oduc ion can occu in ae a ed soil samples i
hey a e incuba ed o long pe iods (> 100 days) unde anoxic condi ions (e.g., Küsel
and D ake 1996; Sexs one and Mains 1990; Vo e al. 2003). Howe e , me hanogens
canno u ilize al e na i e elec on accep o s. Thus, he p ima ily oxic na u e o he
in es iga ed soil, he sho incuba ion ime, and he p esence o al e na i e elec on
accep o s do no a o me hanogenesis (Küsel and D ake 1994; Küsel e al. 1999).
Taking all his indings in o accoun , i can be sugges ed ha (i) he deg ada ion
o cellulose and cellulose-de i ed ca bon in ag icul u al soil is independen o he
a ailabili y o O
2
and (ii) ha he ac i i y o saccha ide-u ilizing soil mic oo gansims
al e s he soil edox po en ial.
4.2. Ac i e Saccha ide-Deg ading P oka yo ic Communi ies
Di e en ae obic and anae obic bac e ial communi ies assimila ed [
13
C]-
cellulose and [
13
C]-cellulose-de i ed saccha ides which esul ed in he de ec ion o
48 labeled amily-le el axa (Table 22). The majo i y o hese amilies had low ela i e
abundances (< 3%) and we e likely o mino impo ance o he deg ada ion o
cellulose (4.2.3). In con as , some amily-le el axa comp ised highe ela i e
abundances (> 3%) wha sugges s ha hese g oups a e celluloly ic and
saccha oly ic key axa in he cellulose-linked ca bon low (4.2.1, 4.2.2).
A chaea we e no labeled in he cu en s udy indica ing ha he expe imen al
condi ions did no a ou he ac i i y o a chaeal communi y membe s, and ha
supplemen ed [
13
C]-saccha ides we e no assimila ed by he a chaeal soil
communi y. This is in con as o s udies on en soil and anoxic municipal was e
land ill co e soil (Hambe ge e al. 2008; Li e al. 2009; Wüs e al. 2009).
C ena chaeo a a e common in he en i onmen and can domina e he ammonium-
oxidizing communi y in ag icul u al soil (A a alli e al. 1998; Gub y-Rangin e al.
2010; Leinigne e al. 2006). Mos abundan A chaea in ae a ed soils a e non-
me hanogenic mesophiles o Thauma chaea (Pes e e al. 2011). These mesophilic
o ganisms a e gene ally assumed o be ni i ie s. None heless, he la ges p opo ion
o he di e si y o soil A chaea is physiologically uncha ac e ized (Ba es e al. 2011;
Pes e e al. 2011). In some a chaeal genomes cellulase genes we e de ec ed (Ando
e al. 2002; Baue e al. 1999), bu known celluloly ic A chaea a e ex eme
he mophiles (G aham e al. 2011; Pe e alo a e al. 2005; Robb e al. 2011). Thus, i
is unlikely ha celluloly ic A chaea play an impo an ole in cellulose deg ada ion in
he in es iga ed empe a e soil communi y.
D
ISCUSSION
119
4.2.1. Key Taxa ha Deg aded Saccha ides unde Oxic Condi ions
Al hough ungal g ow h on cellulose shee s was obse ed (da a no shown)
bac e ia con ibu ed o he deg ada ion o cellulose shee s unde oxic condi ions
(3.2.3.2.1). Celluloly ic and saccha oly ic ae obic axa de ec ed in his s udy ha e
been p e iously iden i ied in and isola ed om soil by he use o c ys alline cellulose
(Haicha e al. 2007; Lo e al. 2009; Pa el and Vaughn 1973; Ul ich e al. 2008). In
con as , p oka yo ic communi ies ha deg ade plan o ganic ma e and lea es a e
dissimila o ac i e axa o he cu en s udy (Be na d e al. 2007; Lee e al. 2011;
Tanahasi e al. 2005). These di e ences migh be based on he composi ion o plan
li e . Plan cell walls consis o a lignocellulose complex, in which cellulose is
embedded in lignin, hemicellulose and o he polyme s such as s a ch and p o eins
(Chang 2007; Kuma e al. 2008a; Malhe be and Cloe e 2002). These polyme s as
well as o he ca bonaceous plan -de i a es (e.g., pho osyn he ic p oduc s) a e
subjec ed o mic obial mine aliza ion (Boddy e al. 2007; Nul sch 2000; Six e al.
2004) and yield a b oade di e si y o soil o ganisms han pu e cellulose. Bac e ial
communi ies esponsible o he deg ada ion o [
13
C]-labeled ice callus a e
domina ed by saccha oly ic membe s o Gammap o eobac e ia, Fla obac e ia,
Sphingobac e ia o Acidobac e ia (Lee e al. 2011). Due o he low cellulose con en
o ice callus, ac i e bac e ia inco po a e [
13
C]-ca bon de i ed om wa e soluble
o ganic compounds, bu less om cellulose (Lee e al. 2011). Likewise, ice s aw is
mainly deg aded by Alpha-, Be a-, Gamma-, and Del ap o eobac e ia, Bac e oide es,
Fla obac e ia, Spi ochae a, and Ve ucomic obia (Bas ian e al. 2009; Be na d e al.
2007; Tanahasi e al. 2005).
Ac inobac e ia we e he mos labeled Bac e ia in he s able iso ope p obing
expe imen and had highes abundances compa ed o o he quan i ied saccha ide-
deg ading axa unde oxic condi ions. Ac inobac e ia comp ise he majo i y o known
ae obic celluloly ic isola es (de Boe e al. 2005; Lynd e al. 2002) and a e one o he
bes s udied phyla in he domain Bac e ia (S ackeb and and Schuhmann 2006).
Ac inobac e ia include s ic and acul a i e ae obic species ha o en build
mycelium-like s uc u es, i.e., hyphae (E iskon 1949; Good ellow and Williams 1983;
S ackeb and and Schuhmann 2006; Ven u a e al. 2007). Hyphae- o ming
celluloly ic membe s may ha e an ecological ad an age. By pene a ing cellulosic
ma e ial hey achie e a be e access o a ackable egions and b ing hei cellulases
in close p oximi y o he subs a e (Abdulla and El-Sha ou y 2007; Cha e e al.
2010; E iksson e al. 1990; Lynd e al. 2002). Celluloly ic and saccha oly ic
Ac inobac e ia a e o en iden i ied as pa s o he bac e ial communi y ha is in ol ed
in he ae obic deg ada ion o plan -de i ed ca bon (e.g., Dumo a and K uglo 2009;
Ho s en e al. 1971; Lee e al. 2011; Lo e al. 2009; Pa el and Vaughn 1973;
Tanahasi e al. 2005; Ul ich e al. 2008). In e es ingly, celluloly ic Ac inobac e ia we e
no equen ly labeled in oxic cellulose-supplemen ed ea men s (Table 22, Figu e
A1). Only some sequences a ilia ed wi h Cellulomonas compos i, a acul a i e
celluloly ic bac e ium o he amily Cellulomonadaceae (Kang e al. 2007). This amily
consis s o p ima ily ae obic species ha a e well known o hei abili y o deg ade
c ys alline cellulose (An e al. 2005; Bagna a e al. 1985; Kang e al. 2007; Lo e al.
2009; Pa el and Vaughn 1973; Ul ich e al. 2008). They a e nume ically impo an in
soil and in ol ed in he deg ada ion o ice s aw o he deg ada ion o o he plan
o ganic ma e (Lo e al. 2009; S ackeb and and Keddie 1986; S ackeb and e al.
2006; Tanahasi e al. 2005). The low co e age o he co esponding gene lib a y
(Table 21; 64%) migh accoun o he low ela i e abundance o celluloly ic
Ac inobac e ia ha deg aded c ys alline cellulose unde oxic condi ions. Wi h he
D
ISCUSSION
126
celluloly ic ac i i y is linked o species o Clus e IV and XIV (Uz and Og am 2006).
Anae obic celluloly ic Clos idia can be limi ed o a ange o ca bohyd a es (Li e al.
2009; Lynd e al. 2002), g owing bes on cellulose and cellodex ins, bu showing
educed g ow h on cellobiose and glucose (Ng and Zeikus 1982; Zhang and Lynd
2005). Fo example, Clos idium he mocellum has bioene ge ic bene i s by g ow h
on cellulose-de i ed cellodex ines which is a esul o he lowe ene gy inpu when
cellodex ines a e aken up, and a e hen in acellula ly clea ed as compa ed o up
ake o cellobiose and glucose (Zhang and Lynd 2005). G ow h o he anae obe
Bac e oides cellulosol ens is only suppo ed by cellulose and cellobiose, bu no by
glucose o o he ca bon sou ces (Mu ay e al. 1984). Ae obic celluloly ic species can
also ha e di e en p e e ences owa ds cellulose and soluble suga s. Mola g ow h
yields o Ac ino alea e men ans (Yi e al. 2007) a e highe on cellulose compa ed o
glucose (Bagna a e al. 1987). Ano he poin ha migh accoun o he obse ed
di e ences is he igh a achmen o he cellulosome o celluloly ic anae obes o
cellulose (Baye e al. 1994a; Baye e al. 2006). As a consequence, he elease o
hyd olysis p oduc s, e.g., cellobiose o glucose, is minimal (Baye e al 1994a; Baye
e al. 2006) wha was p esumably g ow h-limi ing o saccha oly ic sa elli e o ganisms
in anoxic cellulose-supplemen ed ea men s. Supplemen a ion o soluble
saccha ides migh ha e u he a o ed saccha oly ic o ganisms a he han
celluloly ic ones. Saccha oly ic bac e ia a e well adap ed o apidly u ilize cellobiose
o glucose (Lynd e al. 2002), bu ce ain celluloly ic species, e.g., Spo ocy ophaga
myxococcoides, need ime o adap a ion o soluble suga s (Sjips ein and Fa haeus
1949). Hence, an ou -compe i ion o celluloly ic axa by saccha oly ic axa in
cellobiose- and glucose-supplemen ed ea men s is likely.
These conclusi e esul s sugges ha (i) a la ge uncul u ed di e si y o soil
Bac e ia was in ol ed in he deg ada ion o cellulose and p oduc s o i s hyd olysis,
and (ii) he ac i e saccha oly ic communi y di e ed phylogene ically om he ac i e
celluloly ic communi y unde and be ween oxic and anoxic condi ions (Hypo hesis 1,
1.7).
4.2.5. Deg ada ion o Cellulose is a S able Communi y Func ion in he
In es iga ed Soil
The dis ibu ion o oxic and anoxic mic ozones in ae a ed soils is highly dynamic
and may change apidly (1.6.1). I is concei able ha bo h he deg ada ion o
cellulose and he ac i e saccha ide-deg ading communi y is impac ed by luc ua ions
o he a ailabili y o O
2
, e.g., a e p ecipi a ion e en s. Howe e , he e was no
appa en delay in he consump ion o supplemen ed saccha ides du ing oxic and
anoxic pe iods (Figu e 30). O he unc ions o he soil mic obial communi y a e also
no dependen on he a ailabili y o O
2
and he edox po en ial. Fo example, al hough
glucose consump ion a es in ae a ed soil can a y in esponse o O
2
, he capaci y o
he mic obial communi y o consume glucose is independen o edox po en ial (Picek
e al. 2000; San ucko a e al. 2004). Also, he a ailabili y o O
2
does no app eciably
a ec ni ogen u no e a es in opical o es soil, e en hough he p esence o
absence o O
2
engages di e en axa o he mic obial communi y ha a e in ol ed in
ni ogen u no e (Pe -Ridge and Fi es one 2005; Pe -Ridge e al. 2006). Likewise,
selec i e ac i a ion o ae obic and anae obic saccha ide-deg ade s unde con as ing
a ailabili ies o O
2
allowed o a con inous deg ada ion o cellulose in his s udy
(Figu e 32). Mic ococcaceae and Cellulomonadaceae we e he axa ha esponded
mos p onounced o he supplemen a ion o saccha ides unde oxic condi ions
(Figu e 32A). A sligh s imula ion was also e iden o Planc omyce aceae and he
D
ISCUSSION
127
new amily ‘Sphingo1-4’ (Bac e oide es) du ing e-ae a ion (Figu e 32D and G).
Clus e I Clos idiaceae and he new Bac e oide es- ela ed axa ‘Sphingo1-4’ and
‘Cellu1-3’ esponded posi i ely unde anoxic condi ions (Figu e 32C, E and G). Thus,
i can be concluded ha he apid me abolic esponse o unc ionally edundan axa,
i.e., axa ha ha e he capaci y o u ilize he same subs a e unde con as ing
en i onmen al condi ions (e.g., oxic and anoxic), con ibu es o he s abili y o a
pa icula unc ion o he mic obial communi y despi e di e ing a ailabili ies o O
2
(Hypo hesis 2, 1.7).
4.3. Di e si y o Me abolic Ac i e bu no [
13
C]-Labeled Bac e ia
Thi y- i e o 83 de ec ed amily le el- axa we e no labeled in s able iso ope
p obing expe imen s (Table 23; 3.2.3.2). RNA s able iso ope p obing was applied,
because i is mo e ad an ageous han DNA o PLFA (phospholipid a y acid) s able
iso ope p obing (Boschke e al. 1998; Mane ield e al. 2002a; Radajewski e al.
2000). RNA is syn hesised a highe a es han DNA, labeling o RNA occu s wi hou
cellula eplica ion, and RNA p o ides sequence in o ma ion ha allows a highe
phylogene ic esolu ion han PLFA analysis (Mane ield e al. 2002a; Mane ield e al.
2002b; Radajewski e al. 2003). Fu he mo e, DNA pe sis s in he en i onmen a e
cell dea h and canno be easily used o de ine he me abolic s a e (i.e., ac i e o
inac i e) o he o ganism i belongs o (Coolen and O e mann 1998). In con as ,
RNA is labile and deg ades apidly a e cellula inac i a ion o cell dea h (K ame
and Singel on 1993; Lee and Kemp 1994). Hence, he RNA-based de ec ion o non-
labeled amilies indica es ha hese axa we e me abolically ac i e (Mengoni e al.
2005; U ich e al. 2008).
The majo i y o non-labeled axa belonged o he phyla Ac inobac e ia (21.1%),
ollowed by li le abundan axa o Del ap o eobac e ia (6.0%), Be ap o eobac e ia
(5.2%) and Alphap o eobac e ia (3.9%), Gammap o eobac e ia (2.0%), Fi micu es
(1.9%), Bac e oide es (1.4%), Acidobac e ia (1.1%), Chlo o lexi (0.5%),
Ve ucomic obia (0.5%), and Fusobac e ia (0.1%, Table 23). De ec ed non-labeled
ac inobac e ial amilies can ae obically and anae obically u ilize a wide ange o
ca bon compounds p esen in he o ganic ac ion o soil (e.g., suga s, s a ch, pec in)
o a e able o g ow au o ophically (Chen e al. 2004; E ushenko and Takeuchi
2006; Jackson e al. 1995; Kim e al. 2007; Kim e al. 2008; Lynd e al. 2002; Lu e al.
2010; S ackeb and and Schumann 2000; Tamu a e al. 1999; Wade e al. 1999).
Celluloly ic ac i i y wi hin non-labeled Ac inobac e ia is only epo ed o
Mic omonospo aceae (Gallaghe e al. 1996; Lynd e al. 2002; Wilson 1992).
Families o he phylum Bac e oide es and o he phylum Del ap o eobac e ia include
membe s o he so-called ‘gliding bac e ia’, i.e., non- lagella ed bac e ia ha a e
highly mo ile due o su ace-associa ed mo emen (Ne and König 2007). Some
gliding bac e ia a e impo an deg ade s o cellulose in soil (Ne and König 2007;
Reichenbach 2006) o ha e a p eda o y li es yle (Ke s e s e al. 2006). Acidobac e ia,
Fi micu es, and Gammap o eobac e ia ep esen also phyla wi h celluloly ic membe s
(Humph y e al. 2003; Kim 1995; Kuma e al. 2008a) ha can be pa s o he umen
ecosys em in which hey p ima ily e men amino and a y acids (Cook e al. 1994;
an Gylswyk 1995; Janssen and O'Fa ell 1999). Celluloly ic Acidobac e ia a e slow-
g owing cons i uen s o he plan -decomposing communi y in Sphagnum pea , bu
we e likely ou -compe ed by mo e e icien u ilize s o plan -de i ed ca bon, e.g.,
Cy ophagceae species (Eichho s e al. 2011; Pank a o e al. 2008; Pank a o e al.
2011).
D
ISCUSSION
128
The emaining amily-le el axa lack desc ibed celluloly ic species (Table 23),
bu some p ocess cellulase genes (Reinhold-Hu ek e al. 1993; Robledo e al. 2008),
o a e pa s o he cellulose-deg ading communi y in soil and cellulose- ed
en ichmen s (e.g., Anae olineaceae, Alcaligenaceae, and ‘Xiphinema o-
bac e iaceae’; Dumo a and K uglo 2009; Haicha e al. 2007; Ishii e al. 2008; Pa el
and Vaughn 1973). I is supposed ha mos o hese amilies ac as sa elli e
o ganisms and seconda y u ilize s o cellulose-de i ed deg ada ion p oduc s
(Dumo a and K uglo 2009; Ho s en e al. 1971; Ishii e al. 2008). Only 30% o he
ibosomal RNA o an o ganism has o be labeled un il i can be de ec ed by RNA
s able iso ope p obing (Jehmlich e al. 2008). The la ge numbe o non-labeled
amilies migh indica e ha hese Bac e ia inco po a ed amoun s o [
13
C]-ca bon o
less han 30% in o hei RNA, assimila ed soil indigenous ca bon, o ed on o he
bac e ia. The la e wo assump ions a e suppo ed by he enhanced u no e o soil
indigenous ca bon as e lec ed in imbalanced ca bon eco e s unde anoxic
condi ions (4.1) and he physiological capabili ies o hese amily-le el axa, which
include p eda ion and saccha ide u iliza ion.
4.4. Di e si y o Ac i e Euka yo es
The capabli iy o deg ade cellulose is also a wide sp ead ai o mic obial
Euka ya (1.3.1) and di e en euka yo ic amilies we e labeled in oxic [
13
C]-cellulose
ea men s (Table 24). Al hough soil ungi a e majo deg ade s o cellulosic biomass
unde oxic condi ions (de Boe e al. 2005; Lynd e al. 2002) no ungal species was
labeled by [
13
C]-cellulose, nei he unde oxic no unde anoxic condi ions. The lack o
ungi unde anoxic condi ions is consis en wi h he p e ious indings ha anae obic
celluloly ic ungi a e well-known om he in es ine o uminan and non- uminan
he bi o es, bu no om soil (Go don and Phillips 1998; Lynd e al. 2002; Teunissen
and DenCamp 1993), and u he indica es ha ungi played no ole unde anoxic
condi ions. Unde oxic condi ions, a niche di e en ia ion o celluloly ic ungi and
celluloly ic bac e ia, which is go e ned by a se o a iable ac o s, e.g., accessibili y
o cellulose ib es, exc e ion o an i ungal compounds by bac e ia, o pH, is likely
(Bas ian e al. 2009; de Boe e al. 2005; Lynd e al. 2002). Plan ma e ial wi h low
lignin con en may be mo e eadily accessed by celluloly ic bac e ia, and ae obic
celluloly ic bac e ia may be mo e compe i i e a neu al pH and high mois u e
con en s (de Boe e al. 2005; Hi oki and Wa anabe 1996). Howe e , he g ow h o
ungi was obse ed in mic ocosms wi h we soil and cellulosic pape shee s (2.3.3.3)
which indica es ha ungi we e ac i e i he expe imen al condi ions a e sui able.
Well-known ungi ha con ibu e o he deg ada ion o cellulosic ma e ial in ae a ed
soil a e ela ed o he phyla Ascomyco a (e.g., Aspe gillus, Fusa ium, T ichode ma),
Basidiomyco a (e.g., Aga icus, C yp ococcus, Phane ochae e), and Muco omyco ina
(e.g., Muco , Rhizopus) (Bas ias e al. 2009; E iksson 1978; Hammel 1997;
He culano e al. 2011; Lynd e al. 2002; Mandels and Reese 1999; Mahmood e al.
2006). I is likely ha soil ungi we e pa icipan s in he consump ion o cellulose in
his s udy, bu he expe imen al condi ions in s able iso ope p obing expe imen s (i.e.,
he nea neu al pH, he pu i y o he cellulose, high mois u e con en , and cons an
shaking o slu ies) ha e a o ed celluloly ic bac e ia bo h unde oxic and anoxic.
Fu he esea ch is needed o esol e he ole o ungi o he deg ada ion o plan -
de i ed saccha ides in he in es iga ed soil.
D
ISCUSSION
129
In con as o ungal species, p o is s we e labeled in oxic cellulose ea men s
(i.e., Eus igama aceae, Bodonidae, Ch ysophyceae, Opis onec idae,
Mallomonadaceae, S amenopiles; Table 24), bu none o he de ec ed axa has
been desc ibed as being celluloly ic (Be ne and Be ne 1993; Hall 1953; Laybou n-
Pa y and Pa y 2000). Celluloly ic p o is s a e equen ly de ec ed in gu ecosys ems
o uminan s and wood- eeding e mi es in which hey ca alyze he b eakdown o
cellulosic plan ma e ial (Boha ie e al. 1990; B eznak and B une 1994; Coleman
1984; Hidaya e al. 1993; Wheele e al. 2007). Fo example, Oph yoscolecidae-
ela ed cilia es a e impo an anae obic deg ade o cellulose in he umen o sheep
(Coleman 1978) and impo an cons i uen s o he celluloly ic euka yo ic communi y
in ca le (Kudo e al. 1990). Taxa de ec ed in he cu en s udy (Table 24) did no
a ilia e wi h species o celluloly ic p o is s known om uminal ecosys ems wha
indica es ha he p ima y oxic na u e o he soil did no a o anae obic p o is s.
Opis honec a- ela ed species a e ae obic he e o ophic ee-li ing cilia es ha occupy
epheme al wa e bodies o mois u e ilms a ound soil pa icles (Bochdansky and
Huang 2010; Laybou n-Pa y and Pa y 2000; Williams and Clamp 2007). Membe s
o his genus a e bac e io o us and o en ound in nu ien ich was ewa e s (E l
2001; Foissne 1978). Bodonidae a e o he mos equen ly de ec ed he e o ophic
lagella es in se e al ecosys ems (Pa e son and Lee 2000; Scheckenbach e al.
2006). Membe s o hese gene a a e equen ly associa ed wi h agg ega es o
o ganic ma e , e.g., in ac i a ed sludge, on which hey g aze e y e ec i ely on
a ached bac e ia (Laybou n-Pa y and Pa y 2000; on de Heyden and Ca alie -
Smi h 2005). Some species, e.g., membe s o he genus Bodo, a e able o su i e
also a low concen a ions o p ey and can he e o e ou compe e o he bac e io o us
p o is s ha need highe bac e ial densi ies o mul iplica ion (Laybou n-Pa y and
Pa y 2000; Sleigh 2000; Zubko 1995). Simila , Ch ysophyceae- ela ed lagella es
p e e bac e ia as ene gy and ca bon sou ce (Ca on e al. 1986; Yubuki e al. 2008).
Ch ysophyceae a e impo an cons i uen s o he ma ine plank on (Th om 1997), bu
can also be ound in soil samples o di e en geog aphical egions (Boenigk e al.
2005). They display an ae obic he e o ophic as well as an au o ophic li es yle o a e
mixo ophs, i.e., hey combine pho oau o ophy wi h he e o ophy (Sleigh 2000).
Membe s o Ch ysophyceae and o he p o is s can be in ol ed in he compos ing o
ice s aw and o cha d g ass, bu hei ole in he deg ada ion p ocess seems o be o
mino impo ance (Cahyani e al. 2004; Lee e al. 2011). The axa Eus igma aceae
(algal s amenopiles) and Leuka achnion (amoeboid s amenopiles) occu in ma ine
and eshwa e habi a s as well as in soils (Ande sen 2004; G an e al. 2009; Hoek
e al. 1995), and include pho oau o ophs, and he e o ophs ha a e osmo ophic
(Be ne and Be ne 1993; G an e al. 2009). Some s amenopiles a e phago ophic
bac e i ou s, i.e., hey eed on li ing and dead bac e ia (Laybou n-Pa y and Pa y
2000; Sleigh 2000). Membe s o he de ec ed euka yo ic amilies equi e mois zones
in soil, bu can also o m cys s du ing pe iods o d yness o nu ien limi a ion (Hall
1953; Findenig e al. 2010; Laybou n-Pa y and Pa y 2000). In wa e -sa u a ed
habi a s pho o ophic mic oeuka yo es a e impo an p ima y p oduce s o biomass
and d i e he ca bon low h ough ma ine and eshwa e ecosys ems (Hall 1953;
Th om 1997). Mos soil lagella es eed on bac e ia o assimila e o ganic compounds
(Ekelund and Rönn 1994; Mu ase e al. 2006; Rönn e al. 2002), bu hei ole o he
deg ada ion o cellulosic ma e ial is la gely un esol ed. I is likely ha he e o ophic
p o is s inco po a ed [
13
C]-ca bon in he expe imen s du ing he inges ion o [
13
C]-
labeled saccha ide-deg ading bac e ia.
D
ISCUSSION
130
4.5.
Pes icides ha e Mino E ec s on he Me abolism o
Cellulose-Deg ading Communi ies
Soil mic oo ganisms in ag icul u al soils may no only be g azed by
bac e io o us p o is s, bu also impai ed by oxici y o pes icides, such as he bicides,
ungicides, insec icides o b eak down p oduc s he eo (Chowdhu y e al. 2008;
Ge ao e al. 2000; Ka ayama and Kuwa suka 1991; Ka ayama e al. 1992;
Wainw igh 1978). The ex ensi e applica ion o pes icides migh al e cellulose-
dependen ca bon low h ough he mic obial communi y. Thus, saccha oly ic and
celluloly ic axa ha we e iden i ied as key o ganisms o he deg ada ion o cellulose
and i s b eakdown p oduc s (2.3.3.3) we e analysed o hei me abolic esponse on
p esence and absence o pes icides (Table 28).
The ungicides Me alaxyl and Chlo o halonil did appa en ly no a ec he
hyd olysis o cellobiose and subsequen consump ion o glucose a concen a ions
10 – 33 old abo e he concen a ions ecommended o ag icul u e use (Figu e 24;
2.3.3.1). Al hough Me alaxyl may inhibi β-glucosidase ac i i y in soil (Monkiedje and
Spi elle 2002; Sukul 2006), and Chlo o halonil educes cellulose deg ada ion in
ae a ed soils (Ka ayama and Kuwa suka 1991; Ka ayama e al. 1992; Suyama e al.
1993b; Suyama e al. 1993a), he saccha oly ic communi y o he in es iga ed soil is
likely insensi i e o bo h ungicides o nega i e e ec s a e no e lec ed in changed
p ocesses (Figu e 24). Me alaxyl and Chlo o halonil can be decomposed by
mic oo ganisms (Fogg e al. 2003; Monkiedje e al. 2003), bu i is unlikely ha hey
we e deg aded du ing he sho incuba ion ime (2 – 4 days) o he expe imen s o
his wo k (2.3.3.1). Hal -li e imes o Me alaxal and Chlo o halonil a e 9 o 66 days
(Table 5). Hence, he ungicides likely can in e ac wi h he mic obes in soil slu ies.
Se e al easons ha accoun o he lack o inhibi ion a such ‘high’ concen a ions o
pes icides a e concei able: (i) Pes icide-sensi i e saccha ide-deg ading ungi we e
no a ou ed unde he expe imen al condi ions and bac e ia we e pes icide-
insensi i e. (ii) So p ion o Me alaxyl and Chlo o halonil o soil pa icles lowe ed hei
bio oxici y due o educed in e ac ions wi h soil mic oo ganisms (Fogg e al. 2003;
Ka ayama e al. 1991; Sigle e al. 2003). (iii) Pa s o he saccha ide-deg ading
communi y we e selec i ely inhibi ed by he pes icides, bu o he unc ionally
edundan and pes icide-insensi i e axa mimicked his e ec . Howe e , i emains
unclea i and how bo h ungicides impac ed he saccha ide-deg ading communi y,
bu he e ec s o Me alaxyl and Chlo o halonil on he deg ada ion o saccha ides
seem o be minimal. In con as , he pes icides Ben azon, MCPA and Nonylphenol
educed he deg ada ion o cellulose and cellulose-de i ed suga s (Figu e 25 –
Figu e 27, Table 28; 3.5).
Ae obic and anae obic hyd olysis o cellobiose was appa en ly no educed by
low concen a ions o Ben azon, MCPA and Nonylphenol (Figu e 25, Table 27),
sugges ing ha hese pes icides ha e minimal e ec s on he ac i i y o β-
glucosidases o endoglucanases (Figu e 2) a in si u- ele an concen a ions. MCPA
ha is applied a ecommended dose does no a ec ei he ca bon dioxide
p oduc ion, o O
2
up ake and N-mine aliza ion in a c opland soil, and ae obic
cellulose deg ada ion is only li le educed e en when MCPA is di ec ly sp ead on
cellulose shee s (G ossba d 1971; Sch öde 1979). Al hough Nonylphenol can also
inhibi cell g ow h o Bac e ia in pu e cul u e and educes he ni i ying-ac i i y in
ac i a ed sludge (Dokianakis e al. 2006; Okai e al. 2000a), i has no impac on
celluloly ic membe s o he bac e ial soil communi y, e.g., Cy ophaga- ela ed species
(Chang e al. 2007).
D
ISCUSSION
131
In acco dance wi h p e ious s udies (Ce nako a e al. 1991; Hseu 2006; Ma sh
e al. 1978; Piu i e al. 2002), he oxic e ec o Ben azon, MCPA, and Nonylphenol
on he deg ada ion o cellulose-de i ed ca bon, and linked p ocesses was dose-
dependen and only e iden a concen a ions a abo e alues ha a e ypical in
pes icide- ea ed c opland soils (Figu e 26, Table 27). The deg ada ion o c ys alline
cellulose was impai ed bo h unde oxic and anoxic condi ions, i.e. Ben azon, MCPA,
and Nonylphenol educed he amoun o deg aded cellulose by abou 40 – 60%
unde oxic condi ions and by mo e han 90% unde anoxic condi ions (Figu e 27,
Table 27), indica ing ha he anae obic celluloly ic communi y was highly sensi i e o
he oxic e ec s o he pes icides. A simila deg ee o inhibi ion has been epo ed o
Oxy luo en, Piclo am, Me olachlo , and Alachlo unde oxic condi ions (Kucha ski
and Wyszkowska 2008; Sahid and Yap 1994; Sahid and Ramil 1995). Applica ion o
hese pes icides may dec ease cellulase ac i i y and cell numbe s o celluloly ic
bac e ia (Sahid and Yap 1994; Sahid and Ramil 1995). Sul osul u on inhibi s he
g ow h o celluloly ic bac e ia and o he unc ional g oups e en a concen a ions ha
a e ypical o ield use (Kucha ski and Wyszkowska 2008). Simila o cellulose-
supplemen ed ea men s (Figu e 27) anae obic p ocesses, especially he
consump ion o glucose, we e s ongly inhibi ed by all pes icides a high
concen a ions (Figu e 26, Table 27). This is in cong uence wi h s udies ha showed
ha Ben azon can lowe he o al mic obial biomass a e applica ion o soil (Piu i e
al. 2002), and can educe ni ogen mine aliza ion, dini ogen ixa ion, soil espi a ion,
and he cul i abili y o celluloly ic soil bac e ia, celluloy ic ungi and Ac inomyce es
(Ah iainen e al. 2003; Ma sh e al. 1978, Ce nako a e al. 1991).
A gene ally inc eased sensi i i y o anae obes is likely, since some ungicides
may educe cellulose decomposi ion and may impai celluloly ic Clos idia as i has
been shown o a looded soil (Ka ayama and Kuwa suka 1991). This is consis en
wi h he lowe p oduc ion o ca bon dioxide and molecula hyd ogen
in anae obic
pes icide-supplemen ed ea men s, and he educ ion o e ic i on o le els simila o
he con ol wi hou pes icide (Figu e 27, Table 27). I is concei able ha Ben azon,
MCPA, and Nonlyphenol impac ed on memb ane- and cy osol-associa ed p o eins,
and hus did no only speci ically inhibi he ac i i y o celluloly ic and saccha oly ic
axa, bu also he ac i i y o anae obes (e.g., i on educe s) ha may consume
e men a ion p oduc s o anae obic cellulose deg ada ion, i.e., o ganic acids and
alcohols. This conclusion co ela es wi h educed ansc ip numbe s o o al soil
Bac e ia and saccha ide-deg ading axa a he end o he expe imen (Figu e 28,
Table 28). I can be specula ed ha impai men o anae obic p ocesses in well-
ae a ed soils is o mino impo ance o he deg ada ion o cellulose since his
p ocess is mainly ca alyzed by ae obic o ganisms in oxic zones. In pe manen ly
anoxic soil, e.g., looded ice ields, pes icides may ha e s onge impac s on he
ac i e mic obial communi y and may signi ican ly inhibi he cellulose-linked ca bon
low.
De elopmen o p ime s ha a e speci ic o Clus e III Clos idiaceae ailed
(Table 26, 3.4), bu Clus e I Clos idiaceae we e inhibi ed by pes icides. A majo
p opo ion o known anae obic celluloly ic bac e ia ha ha e been isola ed belong o
Clus e III Clos idiaceae. Thus, i canno be excluded ha membe s o Clus e III
Clos idiaceae we e also impai ed and con ibu ed o he e ec o educed
deg ada ion o c ys alline cellulose. Howe e , he saccha oly ic axon
Mic ococcaceae and he celluloly ic axon Cellulomonadaceae we e appa en ly no
in luenced by he applica ion o pes icides since hei RNA con en was no lowe ed
a he end o he incuba ion (Table 28). Ac inobac e ia a e well known o hei
D
ISCUSSION
132
insensi i i y o xenobio ics and hei po en ial o deg ade a wide ange o pes icides,
e.g., benzoni ile he bicides and pa a-ni ophenol (Chike e e al. 2009; Nielsen e al.
2011; Vesela e al. 2010), and hus, may ha e con ibu ed o he deg ada ion o
cellulose despi e he p esence o pes icides. The capabili y o some Bac e ia o
deg ade xenobio ics (e.g., Ekelund e al. 1993; Hseu 2006; Po e and Hayden 2002)
is u he e lec ed in he apid dec ease o Nonylphenol unde oxic condi ions (Figu e
27). Al hough no deg ada ion p oduc s o Nonylphenol we e de ec ed, he p oduc ion
o ca bon dioxide was less educed in Nonylphenol-supplemen ed ea men s han in
he o he pes icide-supplemen ed ea men s (Figu e 27, Table 27). This sugges s
ha ca bon dioxide ha esul s om he deg ada ion o Nonylphenol masked he
impac o Nonylphenol on ca bon dioxide p oduc ion ha esul s om deg ada ion o
cellulose. In e es ingly, g ow h o ungi on cellulose shee s was obse ed (da a no
shown) ha sugges s ha celluloly ic ungi we e insensi i e o he pes icide oxici y,
as i was shown in p e ious s udies (Gi landa e al. 2009; Ko ole a e al. 2002;
Pampulha e al. 2007). Howe e , he impac o pes icides on ungi in he cu en
s udy emains specula i e.
Exposu e o he soil o ganisms o high concen a ions o pes icides is possible
al hough when pes icides a e applied a ecommended a e. He e ogeneous
dis ibu ion o pes icides in c op ields may lead o local maxima o pes icide
concen a ion in soil ha exceed epo ed mean alues (Ma sh e al. 1978). Plan s
can accumula e pes icides in hei issue (Boke n and Ha ms 1997; Po e and
Hayden 2002). Likewise, celluloly ic mic oo ganisms ha use hese pes icide- ea ed
plan s as ca bon and ene gy sou ce can be exposed o highe concen a ions as i
was o iginally applied. Ne e heless, cu en and p e ious da a sugges (i) ha he
anae obic saccha ide-deg ading communi y is mo e sensi i e o he oxici y o
pes icides han he ae obic one and (ii) ha he impac o Ben azon, MCPA and
Nonylphenol on he saccha ide-deg ading communi y is minimal a in si u– ele an
concen a ions (Hypo hesis 3, 1.7).
4.6. Conclusions and Model
The esul s o expe imen s ha we e conduc ed wi hin he doc o al p ojec
illus a e he complexi y o he cellulose-linked ood web. P oka yo ic and euka yo ic
axa ha a e ac i ely in ol ed in he u no e o ca bon in soil slu ies o an ae a ed
ag icul u al soil we e iden i ied. Cellulose and cellulose-de i ed saccha ides a e
u ilized by a highly di e se ae obic and anae obic mic obial communi y, o which a
la ge p opo ion was ep esen ed by new celluloly ic and saccha oly ic species
(Hypo hesis 1, 1.7; Figu e 34). Fluc ua ions o he a ailabili y o O
2
did no impac on
he capabili y o soil mic obial communi y o consume supplemen ed ca bon
compounds (Hypo hesis 2, 1.7; Figu e 34).
Ag icul u al soils a e o en ea ed wi h pes icides, which migh ha e impac s on
he ae obic and anae obic deg ada ion o plan -de i ed saccha ides and he in ol ed
mic obial communi y (Hypo hesis 3, 1.7; Figu e 34). The esul s o he p esen ed
expe imen s do no suppo his hypo hesis. Tes ed pes icides did no impac on he
deg ada ion o saccha ides a in si u- ele an concen a ions. I is possible ha single
saccha ide-deg ading axa we e impac ed by pes icides, pa icula anae obes, bu
his e ec appa en ly plays no ole o he capaci y o he soil mic obial communi y o
deg ade he supplemen ed saccha ides.
D
ISCUSSION
133
Figu e 34. Concep ual model o p ocesses ela ed o he deg ada ion o
cellulose, majo celluloly ic and saccha oly ic bac e ial axa, and he impac o
pes icides in he p esence and absence o O
2
in an ae a ed ag icul u al soil
unde expe imen al condi ions.
The g ay shaded boxes in he lowe po ion o he
model illus a e di e en e en s ha occu du ing he con e sion o saccha ides o p oduc s.
The ela i e amoun s o deg ada ion p oduc s a e indica ed by di e en on sizes. P oposed
names o bac e ial axa in quo a ion ma ks ep esen new amily-le el axa wi hou cul u ed
ep esen a i es (Table 22). Bac e ial axa in b acke s ha e no been p e iously shown o be
celluloly ic, bu we e labeled in cellulose-supplemen ed ea men s. The ape ed box o
edox po en ial indica es ha he edox po en ial became mo e nega i e in he anoxic pe iod.
The ape ed box o high concen a ions o pes icides indica es ha high concen a ions only
inhibi ed he u iliza ion o saccha ides unde anoxic condi ions.
Low [Pes icide]: No E ec
+ O
2
CO
2
Cellulose
Cellobiose
Glucose
Mic ococcaceae
In aspo angiaceae
Mic ococcaceae
‘Sphingo‘ (Bac e oide es)
[‘Deha‘ (Chlo o lexi)]
[Planc omyce aceae]
Consump ion o Cellulose, Cellobiose, and Glucose
Fe men a ions and
Reduc ion o NO
3-
and Fe
3+
Ae obic Respi a ion
CO
2
, H
2
, Ace a e,
P opiona e
CO
2
, H
2
,Ace a e,
P opiona e, Bu y a e,
Lac a e, Isobu y a e,
Succina e
‘Sphingo‘ (Bac e oide es)
‘Cellu‘ (Bac e oide es)
Clos idiaceae I
‘Sphingo‘ (Bac e oide es)
[Kineospo iaceae]
‘Cellu‘ (Bac e oide es)
Clos idiaceae III
Clos idiaceae I
‘High‘
Hyd olysis
High [Pes icide]
(-)
Redox Po en ial
(+)
Hyd olysis
– O
2
D
ISCUSSION
134
Ag icul u al soils a e usually well-ae a ed soils. None heless, oxic and anoxic
mic ozones co-occu (1.6) in which ae obic and anae obic mic obial ac i i ies ake
place (Figu e 3). The collec i e esul s o ae obic and anae obic deg ada ion
p ocesses show ha cellulose, cellobiose, and glucose a e mine alized o ca bon
dioxide by ae obic espi a ion unde oxic condi ions and ha he cellulose, cellobiose,
and glucose a e con e ed o e men a ion p oduc s, concomi an wi h he appa en
educ ion o ni a e and e ic i on unde anoxic condi ions (Figu e 11, Figu e 25 –
Figu e 27, Figu e 30). Ace a e is a dominan e men a ion p oduc du ing anae obic
deg ada ion, ollowed by p opiona e, bu y a e and aces o lac a e, isobu y a e and
succina e. The de ec ed p oduc spec a co ela e well wi h he iden i ica ion o
di e en ae obic and anae obic celluloy ic and saccha oly ic bac e ial axa and hei
known me abolic capabili ies (Table 22; 4.2, 4.2.2). [
13
C]-cellulose is mainly deg aded
by Clus e III Clos idiaceae, and new amily-le el axa wi hin Bac e oide es unde
anoxic condi ions, whe eas new amily-le el axa o he phyla Bac e oide es and
Chlo o lexi a e key deg ade s unde oxic condi ions. Ac i e sub-communi ies in [
13
C]-
cellobiose and [
13
C]-glucose ea men s di e om cellulose deg ading communi ies,
and a e domina ed by Clus e I Clos idiaceae unde anoxic condi ions, whe eas
In aspo angiaceae and Mic ococcaceae a e p e alen in oxic ea men s. In addi ion,
he new amily-le el axa ‘Sphingo1-4’ and ‘Cellu1-3’ we e s imula ed by he addi ion
o cellobiose du ing he anoxic pe iod unde luc ua ing a ailabili ies o O
2
(Figu e
32E and G). Howe e , he ole o o he ac i e axa, i.e., Kineospo iaceae,
Planc omyce aceae, and he new amily-le el axon ‘Deha1’, was no ully esol ed.
These axa likely pa icipa e in he deg ada ion o plan -de i ed saccha ides in he
in es iga ed soil, bu i is s ill unclea a which le el hey a e in ol ed in he cellulose-
linked ood web.
In summa y, Clus e III Clos idiaceae and Bac e oide es- ela ed axa
ep esen ed he majo celluloly ic cons i uen s o he mic obial communi y in slu ies
o he in es iga ed ag icul u al soil, whe eas Clus e I Clos idiaceae, and he
ac inobac e ial amilies Mic ococcaceae and In aspo angiaceae we e saccha oly ic
sa elli e o ganisms ha u ilized cellulose-de i ed hyd olysis p oduc s. The apid
esponse o hese mic obial axa al e ed he edox po en ial du ing he deg ada ion o
saccha ides (Figu e 30), bu he a e a which plan -de i ed saccha ides a e
deg aded was la gely independen o he a ailabili y o O
2
. Selec i e ac i a ion o
unc ionally edundan axa ha coexis in oxic and anoxic mic ozones o ae a ed
ag icul u al soil likely makes he deg ada ion o cellulose a s able unc ion e en when
en i onmen al ac o s change apidly. Pes icides did appa en ly no impai he ac i i y
o he cellulose deg ading-communi y a in si u- ele an concen a ions o pes icides.
The anae obic bac e ial communi y seemed sensi i e o pes icides, bu signi ican
e ec s a e only obse ed a e y high concen a ions ha a e unlikely o occu in
ag icul u al soil.
4.7. Limi a ion o he Applied Me hods
RNA s able iso ope p obing was used as a me hod o selec o bac e ial axa
ha ac i ely consumed [
13
C]-cellulose and cellulose-de i ed hyd olysis p oduc s, i.e.,
cellobiose and glucose, in expe imen s pe o med wi h soil om an ag icul u al
ecosys em. Al hough hese saccha ides we e applied a concen a ions g ea e han
maximum concen a ions de ec ed in soils (Hill e al. 2008; Medei os e al. 2006),
hey ep esen a comp omise be ween low in si u concen a ions and concen a ions
ha a e needed o de ec able inco po a ion o [
13
C] in he RNA pool. The choice o
D
ISCUSSION
135
g adien ac ions ha a e ep esen a i e o labeled and unlabeled RNA was based
on he dis ibu ion o RNA in he g adien (Figu e 13). The ‘hea y’ ac ions a e
en iched o [
13
C]-RNA, bu i is likely ha hey con ain unlabeled RNA (Mane ield e
al. 2002a; Mane ield e al. 2002b; Radajewski e al. 2003). To ensu e ha de ec ed
axa o hese ‘hea y’ ac ions we e iso opically labeled, RFLP analyses and he
cons uc ion o gene lib a ies we e addi ionally applied. I canno be excluded ha he
s udy missed single celluloly ic and saccha oly ic soil axa due o he cons ic i e ype
o da a analysis. Some RFs could no be iden i ied and some ac i e axa migh ha e
also been p esen in non-analysed ac ions. Ne e heless, he iden i ica ion o 48
ac i e amily-le el axa and he dominance o well-cha ac e ized celluloly ic and
saccha oly ic mic oo ganisms (e.g., Clus e I and III Clos idiaceae o
Mic ococcaceae) in RFLP p o iles and gene lib a ies (Table 22; 3.2.3.2.1, 3.2.3.2.2)
indica es ha he majo i y o he cellulose-deg ading communi y was co e ed by he
applied way o da a analysis.
I was possible o moni o communi y dynamics du ing he deg ada ion o
saccha ides (Table 19, Table 20). Al hough labeling o some axa a a la e ime poin
due o assimila ion o [
13
C]-ca bon dioxide canno be excluded, c oss- eeding should
ha e been minimal as he gaseous p oduc s we e pe iodically emo ed and g ow h
o pho oau o ophic o ganisms was p e en ed by da k incuba ion. Taxa ha we e
labeled a la e ime poin s in cellulose ea men s migh ha e needed longe o he
compe i i e u iliza ion o cellulose o migh ha e u ilized soluble hyd olysis p oduc s
om cellulose, e.g., suga s o a y acids (Baye e al. 2006). The deg ada ion o
[
13
C]-en iched whea s aw, ice callus, o cellulose by ag icul u al soil communi ies
also e eals [
13
C]-labeling o mic obial axa a e p olonged incuba ion o which is no
known i hey ep esen celluloly ic axa (Be na d e al. 2007; Lee e al. 2011; Li e al.
2009). S able iso ope p obing can shed ligh on mic obial ood webs in a ious
habi a s (e.g. Hambe ge e al. 2008; Hunge e al. 2011; Mu ell and Radajewski
2000; Wüs e al. 2009), bu c oss- eeding complica es in e p e a ion o labeling
pa e ns, especially o newly disco e ed axa wi h unknown me abolic po en ials.
The e o e, he labeling o known and new celluloly ic and saccha oly ic axa was
quali ied by axon-speci ic qPCR app oaches in he cu en s udy (3.4).
The deg ada ion o CMC does no necessa ily equi e endoglucanases wha
makes CMC a subs a e ha can also be used by saccha oly ic o ganisms (Bisa ia
and Ghose 1981). Fo example, Pan oea species lack endoglucanse-encoding genes
and u ilizes CMC, bu no c ys alline cellulose (Adams e al. 2011). Howe e , he
deg ee o subs i u ion (DS), i.e., he numbe o hyd oxyl g oups ha a e es e i ied
wi h ca boxyme hyl g oups pe glucose uni , o he used CMC was high
(i.e., 0.65 - 0.9). The highe he DS o CMC is, he mo e di icul he subs a e is o
deg ade and he mo e simila i is o c ys alline cellulose (Reese e al. 1950).
The e o e, he used o m o CMC was a sui able analog o c ys alline cellulose.
Taking all hese conside a ions in o accoun i can be concluded ha he iden i ied
celluloly ic and saccha oly ic axa a e likely key mic oo ganism o he deg ada ion o
cellulose and cellulose-de i ed ca bon in he in es iga ed ag icul u al soil.