I
Me abolic Pa hways o
Amino Acids, Monosaccha ides and O ganic Acids
in Soils
assessed by Posi ion-Speci ic Labeling
Disse a ion
zu E langung des G ades
Dok o de Na u wissenscha en
(D . e . na .)
an de Fakul ä Biologie / Chemie / Geowissenscha en
de Uni e si ä Bay eu h
o geleg on
Michaela A. Dippold
(Dipl. Geoökologin & Dipl. Biochemike in)
geb. am 10.07.1982 in Bambe g
Be eue : P o . D . Yako Kuzyako
Bay eu h, den 11. Sep embe 2013
II
Die o liegende A bei wu de in de Zei on 01.03.2010 bis 11.10.2013
in Bay eu h am Leh s uhl ü Ag a ökosys em o schung un e de
Be euung on He n P o . D . Yako Kuzyako ange e ig .
Volls ändige Abd uck de on de Fakul ä ü Biolgie, 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 wis-
senscha en (D . e . na .).
Disse a ion einge eich am: 11.09.2013
Zugelassen du ch die P ü ungskommission: 18.09.2013
Wissenscha liches Kolloquium: 31.01.2014
Am ie ende Dekan: P o . D . Rhe Kempe
P ü ungsauschuss:
P o . D . Y. Kuzyako (E s gu ach e )
P o . D . C. We ne -Pin o (Zwei gu ach e )
P o . D . E. Ma zne (Vo si z)
PD D . M. Ho n
D i gu ach e : PD D . G. Wiesenbe g
Con en s
I
I. Con en s
I.
Con en s ............................................................................................... I
II.
Lis o Figu es....................................................................................IX
III.
Lis o Tables ................................................................................... XV
IV.
Abb e ia ions................................................................................. XVII
V.
Summa y ....................................................................................... XVIII
VI.
Zusammen assung.......................................................................... XX
1
Ex ended Summa y.............................................................................................. 1
1.1
In oduc ion ...................................................................................................... 1
1.1.1
Low molecula weigh o ganic subs ances in soils.......................................... 1
1.1.1.1
Role and ele ance o low molecula weigh o ganic subs ances in soil...1
1.1.1.2
Sou ces and sinks o LMWOS.................................................................2
1.1.2
Mic obial u iliza ion o low molecula weigh o ganic subs ances.................... 4
1.1.2.1
Mic obial up ake o LMWOS: he mos compe i i e p ocess de e mining
he a e o LMWOS-C in soil ....................................................................4
1.1.2.2
Mine aliza ion e sus inco po a ion o LMWOS-C in o mic oo ganisms...5
1.1.3
Me abolic acing by posi ion-speci ic labeling ................................................ 6
1.1.4
Objec i es ...................................................................................................... 8
1.2
Expe imen s and Me hods............................................................................... 8
1.2.1
Field expe imen ............................................................................................. 8
1.2.2
Labo a o y expe imen s.................................................................................10
1.2.2.1
Expe imen 1: T ans o ma ions o ee alanine.......................................10
1.2.2.2
Expe imen 2: T ans o ma ions o so bed alanine..................................11
1.2.2.3
Expe imen 3: Plan up ake o in ac alanine..........................................11
1.2.3
Me hods o ace
13
C and
14
C in ans o ma ion p oduc s o LMWOS.............11
1.2.3.1
Bulk-iso ope measu emen s by EA-IRMS..............................................12
1.2.3.2
Compound-speci ic iso ope analysis o mic obial bioma ke s ................12
1.2.3.3
Radiochemical analyses........................................................................13
1.2.4
The Di e gence Index ...................................................................................14
1.3
Resul s and Discussion..................................................................................15
1.3.1
O e iew: main esul s o he s udies ............................................................15
Con en s
II
1.3.2
De e mina ion o me abolic pa hways o amino acids, monosaccha ides and
o ganic acids.................................................................................................16
1.3.2.1
Simila i ies and di e ences o indi idual LMWOS..................................16
1.3.2.2
The main pa hways o LMWOS me aboliza ion by soil mic oo ganisms19
1.3.2.3
Me abolic pa hways o he o ma ion o speci ic cellula compounds ....22
1.3.3
Iden i ica ion o speci ic me abolic pa hways..................................................24
1.3.3.1
Speci ic pa hways o indi idual membe s o he mic obial communi y in
soils.......................................................................................................24
1.3.3.2
Pa hways unde a ious concen a ions o LMWOS ..............................25
1.3.3.3
Pa hways o so bed LMWOS.................................................................27
1.3.3.4
Ex a- e sus in acellula ans o ma ion pa hways...............................29
1.3.4
Kine ics and ecological ele ance o compe ing sinks o LMWOS................30
1.3.4.1
So p ion e sus mic obial u iliza ion.......................................................30
1.3.4.2
Plan up ake e sus mic obial u iliza ion ................................................30
1.4
Conclusions.....................................................................................................32
1.5
Re e ence Lis .................................................................................................35
1.6
Con ibu ion o he included manusc ip s and publica ions........................40
2
Publica ions and Manusc ip s............................................................................44
2.1
S udy 1: Fa e o low molecula weigh o ganic subs ances in an a able soil:
om mic obial up ake o u ilisa ion and s abilisa ion ..................................44
Abs ac ....................................................................................................................45
2.1.1
In oduc ion ...................................................................................................47
2.1.2
Ma e ial and Me hods....................................................................................49
2.1.2.1
Expe imen al design..............................................................................49
2.1.2.2
Bulk soil δ
13
C analysis ...........................................................................50
2.1.2.3
Mic obial biomass..................................................................................50
2.1.2.4
Phospholipid a y acid analysis.............................................................51
2.1.2.5
Calcula ions and s a is ical analysis.......................................................52
2.1.3
Resul s..........................................................................................................53
2.1.3.1
Mic obial communi y s uc u e ...............................................................53
2.1.3.2
Mic obial u ilisa ion o LMWOS..............................................................54
2.1.3.3
U ilisa ion o LMWOS by unc ional mic obial g oups.............................55
2.1.4
Discussion.....................................................................................................59
2.1.4.1
Inco po a ion o LMWOS in o SOM and mic obial biomass....................59
2.1.4.2
Mic obial communi y composi ion..........................................................62
2.1.4.3
Inco po a ion o LMWOS in o PLFAs.....................................................63
Con en s
III
2.1.5
Conclusion ....................................................................................................66
Acknowledgemen s...................................................................................................67
Re e ences ...............................................................................................................68
Supplemen a y Da a.................................................................................................72
2.2
S udy 2: Imp o ed δ
13
C analysis o amino suga s in soil by Ion
Ch oma og aphy – Oxida ion – Iso ope Ra io Mass Spec ome y .............74
Abs ac ....................................................................................................................75
2.2.1
In oduc ion ...................................................................................................76
2.2.2
Ma e ial and Me hods....................................................................................78
2.2.2.1
Soil ........................................................................................................78
2.2.2.2
Chemicals, eagen s and ex e nal and in e nal s anda ds......................78
2.2.2.3
Soil hyd olysis and ion emo al .............................................................79
2.2.2.4
Pu i ica ion by ca ion exchange column.................................................79
2.2.2.5
De elopmen o he measu emen by IC-O-IRMS..................................79
2.2.2.6
E alua ion o amino suga quan i ica ion ia IC-O-IRMS........................81
2.2.2.7
E alua ion o δ
13
C de e mina ion ia IC-O-IRMS....................................82
2.2.3
Resul s and Discussion .................................................................................83
2.2.3.1
Ch oma og aphy....................................................................................83
2.2.3.2
Reco e y, linea i y, p ecision and de ec ion and quan i ica ion limi s .....84
2.2.3.3
3.3 Amoun dependence and co ec ion ac o s o δ
13
C alues..............85
2.2.3.4
3.4 Accu acy, p ecision and iso opic LoQ o δ
13
C de e mina ion............86
2.2.3.5
Ad an ages o IC-O-IRMS.....................................................................89
2.2.4
Conclusions...................................................................................................89
Acknowledgmen s.....................................................................................................90
Re e ence Lis ...........................................................................................................91
Supplemen a y Da a.................................................................................................93
2.3
S udy 3: Biochemical pa hways o amino acids in soil: E alua ion by
posi ion-speci ic labeling and
13
C-PLFA analysis.........................................96
Abs ac ....................................................................................................................97
2.3.1
In oduc ion ...................................................................................................98
2.3.2
Ma e ial and Me hods..................................................................................100
2.3.2.1
Field expe imen ..................................................................................100
2.3.2.2
Analy ical me hods...............................................................................101
2.3.2.3
Di e gence Index.................................................................................104
2.3.2.4
S a is ical analysis ...............................................................................105

Con en s
IV
2.3.3
Resul s........................................................................................................105
2.3.3.1
Inco po a ion o uni o mly labeled amino acids....................................105
2.3.3.2
Inco po a ion o posi ion-speci ically labeled amino acids....................106
2.3.3.3
Di e gence Index.................................................................................108
2.3.4
Discussion...................................................................................................110
2.3.4.1
Inco po a ion o ca bon om amino acids in soil and mic obial biomass
110
2.3.4.2
Inco po a ion o ace in o he mic obial g oups ..................................111
2.3.4.3
Disc imina ion o indi idual ca bon posi ions by mic obial u iliza ion di e s
depending on oxida ion s a e, amino acid and ime..............................113
2.3.5
Conclusions.................................................................................................115
Acknowledgemen s.................................................................................................116
Re e ence Lis .........................................................................................................117
Supplemen a y Da a...............................................................................................120
2.4
S udy 4: Biogeochemical ans o ma ions o amino acids in soil assessed
by posi ion-speci ic labeling ........................................................................122
Abs ac ..................................................................................................................123
2.4.1
In oduc ion .................................................................................................124
2.4.2
Ma e ial and Me hods..................................................................................126
2.4.2.1
Soil ......................................................................................................126
2.4.2.2
Chemicals and adiochemicals ............................................................126
2.4.2.3
Expe imen al se up..............................................................................126
2.4.2.4
Radiochemical analyses......................................................................129
2.4.2.5
Calcula ion o he kine ics o alanine u iliza ion ....................................129
2.4.2.6
Calcula ion o he dis ibu ion o alanine-C in ans o ma ion p oduc s.130
2.4.2.7
S a is ics..............................................................................................131
2.4.3
Resul s........................................................................................................132
2.4.3.1
E alua ion o esul s quali y .................................................................132
2.4.3.2
So p ion o alanine o he soil ma ix....................................................133
2.4.3.3
Kine ics o bio ic alanine u iliza ion.......................................................134
2.4.3.4
Bio ic ans o ma ion p oduc s o alanine.............................................136
2.4.3.5
Posi ion-speci ic di e ences o he alanine ans o ma ion pa hways...137
2.4.4
Discussion...................................................................................................138
2.4.4.1
So p ion o alanine occu s as a whole molecule ..................................138
2.4.4.2
Kine ics o ex acellula ans o ma ion and mic obial up ake...............139
2.4.4.3
Exoenzyma ic ans o ma ion p oduc s................................................140
Con en s
V
2.4.4.4
Me abolic pa hways and hei in acellula ans o ma ion p oduc s .....141
2.4.5
Conclusions and Ou look.............................................................................143
Acknowledgemen ..................................................................................................144
Re e ence Lis .........................................................................................................145
Supplemen a y Da a...............................................................................................148
2.5
S udy 5: So p ion a ec s amino acid pa hways in soil: Implica ion om
posi ion-speci ic labeling o alanine............................................................151
Abs ac ..................................................................................................................152
2.5.1
In oduc ion .................................................................................................154
2.5.2
Ma e ial and Me hods..................................................................................156
2.5.2.1
Soil ......................................................................................................156
2.5.2.2
So ben s..............................................................................................156
2.5.2.3
Chemicals and adiochemicals ............................................................157
2.5.2.4
P e-expe imen s ..................................................................................157
2.5.2.5
Expe imen al Se up .............................................................................158
2.5.2.6
Chemical and adiochemical analyses.................................................159
2.5.2.7
Calcula ions and modeling...................................................................159
2.5.2.8
Calcula ion o he C-1/C-2,3- a io and he Di e gence Index DI
i
..........160
2.5.2.9
S a is ics..............................................................................................161
2.5.3
Resul s........................................................................................................162
2.5.3.1
So p ion and mic obial u iliza ion o uni o mly labeled alanine .............162
2.5.3.2
Kine ics o posi ion-speci ic u iliza ion o so bed alanine C...................164
2.5.3.3
Inco po a ion o C om alanine posi ions in s abilized pools and
decomposi ion o CO
2
..........................................................................167
2.5.4
Discussion...................................................................................................169
2.5.4.1
So p ion mechanisms o amino acids ..................................................169
2.5.4.2
Bioa ailabili y o so bed alanine...........................................................172
2.5.4.3
Pa hways o mic obial me aboliza ion o so bed alanine......................173
2.5.4.4
S abiliza ion o amino acid C by so p ion .............................................176
2.5.5
Conclusions and Ou look.............................................................................177
Acknowledgmen s...................................................................................................178
Re e ence Lis .........................................................................................................179
Supplemen a y Da a...............................................................................................183
2.6
S udy 6: Biochemis y o hexose and pen ose ans o ma ions in soil
analyzed by posi ion-speci ic labeling and
13
C-PLFA.................................184
Abs ac ..................................................................................................................185
Con en s
VI
2.6.1
In oduc ion .................................................................................................186
2.6.2
Ma e ial and Me hods..................................................................................188
2.6.2.1
Sampling Si e ......................................................................................188
2.6.2.2
Analy ical me hods...............................................................................189
2.6.2.3
Di e gence Index.................................................................................192
2.6.2.4
S a is ical analysis ...............................................................................192
2.6.3
Resul s........................................................................................................193
2.6.3.1
Inco po a ion o uni o mly labeled monosaccha ides ...........................193
2.6.3.2
Inco po a ion o posi ion-speci ically labeled monosaccha ides ...........193
2.6.3.3
T ace up ake o unc ional mic obial g oups........................................195
2.6.3.4
Di e gence Index.................................................................................196
2.6.4
Discussion...................................................................................................196
2.6.4.1
Glucose and Ribose inco po a ion in o soil and mic obial biomass......196
2.6.4.2
Mic obial u iliza ion o indi idual posi ions o glucose and ibose
molecules ............................................................................................198
2.6.4.3
Speci ic pa hways o glucose and ibose u iliza ion by indi idual mic obial
g oups .................................................................................................199
2.6.4.4
Me abolic acing by posi ion-speci ic labeling o monosaccha ides.....202
2.6.5
Conclusions and Ou look.............................................................................203
Acknowledgemen s.................................................................................................204
Re e ences .............................................................................................................205
Supplemen a y Da a...............................................................................................208
2.7
S udy 7: Me abolic pa hways o ungal and bac e ial amino suga o ma ion
in soil assessed by posi ion-speci ic
13
C-labeling ......................................210
Abs ac ..................................................................................................................211
2.7.1
In oduc ion .................................................................................................213
2.7.2
Ma e ial and Me hods..................................................................................215
2.7.2.1
Expe imen al Si e ................................................................................215
2.7.2.2
Expe imen Design ..............................................................................215
2.7.2.3
Sampling and Sample P epa a ion ......................................................216
2.7.2.4
Bulk Soil and Mic obial Biomass Analysis............................................216
2.7.2.5
Amino suga δ
13
C analysis...................................................................217
2.7.2.6
Di e gence Index.................................................................................219
2.7.2.7
S a is ics..............................................................................................220
2.7.3
Resul s........................................................................................................220
2.7.3.1
Glucose
13
C inco po a ion in o soil and mic obial C pools....................220
Con en s
VII
2.7.3.2
Inco po a ion o C om a ious posi ions o glucose molecule in o
indi idual amino suga s .......................................................................221
2.7.3.3
Replacemen o cell wall pool by glucose
13
C.......................................223
2.7.4
Discussion...................................................................................................224
2.7.4.1
Fungal e sus bac e ial con ibu ion o he amino suga inge p in and
glucose u iliza ion ................................................................................224
2.7.4.2
Pa hways o amino suga o ma ion.....................................................225
2.7.4.3
Speci ic pa hways o ungi and bac e ia...............................................228
2.7.5
Conclusions and Ou look.............................................................................229
Acknowledgemen s.................................................................................................230
Re e ences .............................................................................................................231
Supplemen a y Da a...............................................................................................235
2.8
S udy 8: Fo ma ion and ans o ma ion o a y acids in soil assessed by
posi ion-speci ic labeling o p ecu so s......................................................236
Abs ac ..................................................................................................................237
2.8.1
In oduc ion .................................................................................................239
2.8.2
Ma e ial and Me hods..................................................................................241
2.8.2.1
Expe imen al Si e ................................................................................241
2.8.2.2
Expe imen Design ..............................................................................242
2.8.2.3
Sampling and Sample P epa a ion ......................................................242
2.8.2.4
Bulk Soil and Mic obial Biomass Analysis............................................242
2.8.2.5
PLFA δ
13
C analysis..............................................................................243
2.8.2.6
Fa y acid g ouping..............................................................................245
2.8.2.7
The Di e gence Index DI
i
.....................................................................245
2.8.2.8
S a is ics..............................................................................................246
2.8.3
Resul s........................................................................................................246
2.8.3.1
Inco po a ion o
13
C in soil and mic obial biomass................................246
2.8.3.2
Inco po a ion o C om a ious posi ions o ace a e and palmi a e in o
indi idual PLFAs..................................................................................247
2.8.3.3
Inco po a ion o ace a e and palmi a e
13
C in o PLFAs o indi idual
mic obial g oups ..................................................................................249
2.8.4
Discussion...................................................................................................252
2.8.4.1
U iliza ion and u no e o ace a e and palmi a e by soil mic obial
communi y...........................................................................................252
2.8.4.2
Pa hways o a y acid o ma ion om ace a e in soil............................253
2.8.4.3
Pa hways o a y acid ans o ma ions in soils.....................................254
Lis o Figu es
XIV
Fig. 2
Pe cen age o
14
C inco po a ion in oo s and shoo s a e uni o m
14
C labeling wi h
ace a e and alanine. Le e s indica e he signi ican di e ences (p<0.001) o
ace a e and alanine C be ween plan s ...............................................................278
Fig. 3
Pe cen age o
14
C inco po a ion in oo s and shoo s a e posi ion-speci ic labeling
wi h alanine. The alanine posi ions we e C-1 (ca boxyl g oup), C-2 (amino-bound
g oup) and C-3 (me hyl g oup). Le e s indica e signi ican di e ences (p<0.001)
be ween alanine C posi ions. .............................................................................279
Fig. 4
Ra io o
14
C/
15
N o indi idual alanine C posi ions inco po a ed in plan biomass.
The alanine posi ions we e C-1 (ca boxyl g oup), C-2 (amino-bound g oup) and C-
3 (me hyl g oup). Le e s indica e signi ican di e ences (p<0.001) be ween
alanine C posi ions.............................................................................................280
Fig. 5
Illus a ion o he a e o alanine ace molecules, which a e ei he aken up in ac
o deg aded/mine alized o agmen s and subsequen ly inco po a ed in o plan
biomass o mic oo ganisms. Mic obial me abolism o alanine by mic oo ganisms is
adap ed om Dippold & Kuzyako (2013)..........................................................282

Lis o Tables
XV
III. Lis o Tables
Ex ended Summa y
Table S1
T ea men s o posi ion-speci ic
13
C and
15
N labeling. Applied amoun o
13
C and
15
N, hei iso opic en ichmen , as well as he espec i e compound and labeled
posi ion a e p esen ed. Na . abund. means applica ion o non-en iched
subs ances, x means“no applica ion o
15
N in his ea men . ...............................10
Table S2
Applied
13
C- and
14
C-labeling app oaches as well as analy ical me hods o he
indi idual s udies; Fi s line shows whe he samples we e de i ed om ield o
labo a o y expe imen s; PS indica es posi ion-speci ic labeling............................12
Table S3
Ti le o he indi idual s udies as well as hei objec i es and main conclusions. ...15
Table S4
Analogies in he beha io o indi idual C posi ions o LMWOS en e ing he main
b anch o he basic C me abolism (glycolysis, py u a e dehyd ogenase and ci ic
acid cycle). Analogies we e concluded om he basic me abolic pa hways shown
in Figu e S6. Posi ions wi hin one column a e equi alen wi hin hese pa hways. 21
Publica ions and Manusc ip s:
S udy 1:
Table 1
Absolu e and ela i e abundance (absolu e in µg pe g and ela i e in % o o al
PLFAs) o he a y acids o he mic obial g oups, classi ied by ac o analysis
( ac o loadings see Supplemen a y Table 2).......................................................54
S udy 2:
Table 1
Reco e y (%), ela i e s anda d de ia ion (RSD) and pa ame e s o eg ession
analysis as well as de ec ion (LoD) and quan i ica ion limi s (LoQ) o he
quan i ica ion o amino suga s assessed om he s anda d addi ion expe imen . 85
Table 2
Compa ison o δ
13
C(S d)
EA-IRMS
(EA-IRMS PeeDeeBe calib a ed δ
13
C alue o
s anda d subs ances spiked o he sample) and δ
13
C(S d)
IC-O-IRMS
( i ed δ
13
C alue
o he spiked s anda ds om he mixing model o he s anda d addi ion me hod)
e lec ing he accu acy o IC-O-IRMS measu emen . Fi ed δ
13
C alues o soil
om mixing model (δ
13
C(soil)
calcula ed
) and eal measu emen o un-spiked soil
δ
13
C(soil)
IC-O-IRMS
a e also p esen ed. P ecision is shown 1) by he s anda d
de ia ion o he measu emen epe i ions and 2) by calcula ing he a ea dependen
s anda d de ia ion acco ding o equa ion 6 o he measu ed peak a ea. Iso opic
LoQ e lec s he minimum amoun pe ial needed o ecei e a s anda d e o o
he measu emen epe i ions lowe han 0.5‰. Gal=galac osamin,
Glc=glucosamine and Mu A=mu amic acid..........................................................87
S udy 3:
Table 1
Concen a ions o amino acid solu ions o soil labeling .....................................101
Table 2
To al C con en and
13
C inco po a ion o uni o mly labeled amino acids in o soil,
mic obial biomass and sum o PLFA (Σ-PLFA)...................................................105
S udy 4:
Table 1
Pa ame e s o he Michaelis-Men en kine ics o ea men s wi h inhibi ion o
espi a ion (eq. 2) and ea men s wi hou inhibi ion (eq. 6). R
2
is he coe icien o
de e mina ion and s a s show signi icance o he espec i e non-linea i ing esul
( espec i es cu es a e plo ed in Figu e 4)........................................................136
Lis o Tables
XVI
S udy 5:
Table 1
E ec i e ca ion exchange capaci y and speci ic su ace a ea o he i e so ben s
and he soil used o his expe imen ..................................................................157
Table 2
Ini ially so bed alanine C and i ed pa ame e s o he ou -pool model o mic obial
u iliza ion o so bed alanine (Fig. 1) i ed o he da a o uni o m alanine labeling
..........................................................................................................................162
Table 3
Fi ed pa ame e s o he ou -pool model o mic obial u iliza ion o so bed alanine
(Fig. 1) o he indi idual alanine C posi ions......................................................165
S udy 6:
Table 1
Loca ions o
13
C in posi ion speci ically labeled glucose and ibose and hei
amoun s added o soil in he ield expe imen .....................................................188
Table 2
To al C con en and
13
C inco po a ion o uni o mly labeled monosaccha ides in o
soil, mic obial biomass and sum o PLFA (Σ-PLFA). ..........................................193
S udy 7:
Table 1
Amoun and glucose
13
C eco e y in o al o ganic C (TOC), mic obial biomass C
(C
mic
) and he o al amino suga s (Σ
AminoSuga s
) as well as he h ee indi idual amino
suga s................................................................................................................221
Table 2
Theo e ic C pa e n o newly o med amino suga s a e simple pa hway
combina ions o basic C me abolism..................................................................228
S udy 8:
Table 1
To al o ganic C (TOC), mic obial biomass C (C
mic
) and he sum o all measu ed
PLFAs (lis o a y acids see Supplemen a y, Table A1) in mg C pe g soil (d y
weigh )...............................................................................................................246
S udy 9:
Table 1
The physicochemical p ope ies o he Ap-ho izon o he haplic Lu isol. ............273
Table 2
Shoo / oo a io o
15
N om indi idual N sou ces................................................278
Table 3
In ac up ake o alanine by chico y, lupine and maize and es ima ed con ibu ion o
in ac alanine up ake o o al N nu i ion o hese plan s wi h espec o he o he N
sou ces. .............................................................................................................281
Abb e ia ions
XVII
IV. Abb e ia ions
ANCOVA Analysis o Co a iance
ANOVA Analysis o Va iance
C Ca bon
CEC Ca ion Exchange Capaci y
DI Di e gence Index
DOC Dissol ed O ganic Ca bon
DON Dissol e O ganic Ni ogen
EA Elemen al Analyze
FAME Fa y Acid Me hyl Es e
GC Gas Ch oma og aphy
GC-C-IRMS Gas Ch oma og aphy-Combus ion-Iso ope Ra io Mass
Spec ome e
HPLC High P essu e Liquid Ch oma og aphy
IC Ion Ch oma og aphy
IC-O-IRMS Ion Ch oma og aph-Oxida ion-Iso ope Ra io Mass
Spec ome e
IRMS Iso ope Ra io Mass Spec ome e
IS In e nal S anda d
LMWOS Low Molecula Weigh O ganic Subs ances
MS Mass Spec ome e
N Ni ogen
PEEK Polye he e he ke on
PLFA Phospholipid Fa y Acid
SIM Selec ed Ion Mode
SOC Soil O ganic Ca bon
SOM Soil O ganic Ma e
Summa y
XVIII
V. Summa y
T ans o ma ion o low molecula weigh o ganic subs ances (LMWOS) is one o he
mos impo an s eps in biogeochemical cycles since all high molecula subs ances pass his
s age du ing hei decomposi ion. Mic obial u iliza ion is he mos ele an sink o LMWOS in
soils and hus knowledge abou mic obial ans o ma ions o LMWOS is c ucial o unde -
s anding he soil o ganic ca bon (SOC) cycle and p edic ing i s eac ion o changes in con-
olling en i onmen al pa ame e s. P e ious s udies ocused on de e mining luxes h ough
he LMWOS pool, bu hey a ely iden i ied ans o ma ion s eps. This hesis aims o es ablish
posi ion-speci ic iso ope labeling as a ool in soil science o ace he pa hways o LMWOS
ans o ma ions.
In a medium- e m ield expe imen six posi ion-speci ic
13
C-labeled LMWOS om he
h ee main LMWOS classes we e applied: wo amino acids (alanine and glu ama e), wo
monosaccha ides (glucose and ibose) and wo o ganic acids (ace a e and palmi a e).
13
C
emaining in soil and ha inco po a ed in o mic obial biomass and speci ic mic obial cellula
compounds (phospholipid a y acids (PLFA) and amino suga s) was de e mined by bulk and
compound-speci ic
13
C analyses. The e o e, a new ins umen coupling, an ion ch oma o-
g aph wi h an iso ope a io mass spec ome e (IC-O-IRMS), and he espec i e me hods o
amino suga analysis we e es ablished. The e ec o al e ed en i onmen al condi ions and
he ele ance o u he LMWOS sinks (so p ion o plan up ake) we e e alua ed in se e al
addi ional labo a o y expe imen s based on posi ion-speci ic
14
C-labeling. The di e gence
index (DI) was es ablished o compa e he posi ion-speci ic a e o indi idual subs ances in
a ious s udies independen o he iso opic app oach o expe imen al design used o he pool
in es iga ed.
Mic obial u iliza ion was he as es p ocess in he emo al o LMWOS om soil solu-
ion and nei he plan up ake no so p ion could ou -compe e mic oo ganisms. The inco po a-
ion o indi idual molecule posi ions in soils, mic obial biomass and dis inc compound
classes was clea ly de ined by he mic obial me abolism: Glycolysis, oxida ion by py u a e
dehyd ogenase and he ci ic acid cycle we e iden i ied as he main me abolic p ocesses.
Howe e , in addi ion o hese oxidizing ca abolic pa hways, he anabolic pa hways, i.e. build-
ing-up new cellula compounds, occu ed in soils simul aneously. This in ol ed an in ensi e
C ecycling and u no e wi hin he mic oo ganisms ha was obse ed no only o cy osolic
compounds bu also o cell wall polyme s. In ensi e modi ica ions and ans o ma ion wi hin
me abolic side b anches, like he a y acid o ma ion and ans o ma ion pa hways, we e
iden i ied. These esul s o a y acid ans o ma ions a e c ucial o hei applica ion as plan
bioma ke s in s udies on palaeoen i onmen al econs uc ion.
The combina ion o posi ion-speci ic
13
C-labeling wi h compound-speci ic iso ope
analysis o mic obial bioma ke s allowed he u he iden i ica ion o speci ic pa hways o in-
Summa y
XIX
di idual unc ional mic obial g oups in soils. Fungal me abolism was shown o be slowe han
bac e ial in acellula C ecycling and u no e , which p o ides he me abolic eason o he
slow-cycling ungal-based and as -cycling bac e ia-based b anch o he soil ood web. Shi s
in C alloca ion h ough a ious me abolic pa hways we e dependen on en i onmen al ac-
o s: a g adien o C me abolism om s a a ion pa hways ia main enance me abolism o
me abolic pa hways cha ac e is ic o mic obial g ow h was obse ed wi h inc easing sub-
s a e concen a ion. So p ion, also limi ing he bioa ailabili y o a subs a e, caused simila
shi s in me abolic pa hways: he lowe he bioa ailabili y (e.g. due o so p ion), he mo e C
was alloca ed owa ds anabolic biosyn hesis, i.e. in o mic obial p oduc s. Thus, hese s udies
e ealed ha posi ion-speci ic labeling is no only a aluable ool in biochemis y o me a-
bolic lux analysis, bu also enables he econs uc ion o me abolic pa hways o LMWOS
wi hin di e se mic obial communi ies in complex media such as soil. P ocesses occu ing
simul aneously in soil i.e. 1) wi hin indi idual, e e sible me abolic pa hways, 2) in a ious
mic obial g oups o 3) in speci ic mic ohabi a s (like on mine al su aces, a he soil-plan in-
e ace o a ho -spo s e sus bulk soil) could be aced by posi ion-speci ic labeling in soils in
si u.
The main me abolic pa hways o mic obial LMWOS ans o ma ion by ca a- and anabolism
we e aced by posi ion-speci ic labeling. These pa hways and hei egula ing ac o s a e
c ucial o assessing C lows owa ds mine aliza ion e sus he o ma ion o mic obial bio-
mass, he p e equisi e o he o ma ion o mic obially-de i ed SOC. This molecula knowl-
edge o ans o ma ion s eps and hei egula ing ac o s is c ucial o p edic (i.e. by new
p ocess-based modelling app oaches) and manipula e C alloca ion and s abiliza ion in soils.

Zusammen assung
XX
VI. Zusammen assung
Die T ans o ma ion niede molekula e o ganische Subs anzen (LMWOS) is de zen -
ale Sch i in biogeochemischen K eisläu en, da alle hochmolekula en Subs anzen wäh end
ih es Abbaus den LMWOS Pool passie en. Mik oo ganismen s ellen die bedeu ends e Senke
ü LMWOS da , weshalb mik obielle T ans o ma ionen on LMWOS essen iell ü den Koh-
lens o k eislau im Boden sind. Bishe ige S udien quan i izie en meis Flüsse du ch den
LMWOS Pool, a bei e en abe kaum an de Au klä ung de T ans o ma ionsp ozesse. Im
Rahmen diese Disse a ion soll die posi ionsspezi ische Iso openma kie ung als neue bo-
denkundliche Me hode zu Au klä ung on LMWOS-T ans o ma ionswegen e ablie we den.
In einem Feldexpe imen wu den sechs posi ionsspezi isch
13
C ma kie e LMWOS de
d ei wich igs en Subs anzklassen applizie : zwei Aminosäu en (Alanin und Glu ama ), zwei
Monosaccha ide (Glucose und Ribose) und zwei o ganische Säu en (Ace a und Palmi a ).
Die Analyse on e bleibendem
13
C im Boden,
13
C in de mik obiellen Biomasse und in spe-
zi ischen Zellbaus einen (Phospholipid e säu en (PLFA) und Aminozucke ) e olg e du ch
gesam - und komponen enspezi ische
13
C Me hoden. Hie ü wu de eine neue Ins umen en-
kopplung – ein Ionench oma og aph mi einem Iso openmassenspek ome e (IC-O-IRMS) –
e ablie und die da au abges imm e Aminozucke -Au einingungsme hode eingea bei e .
De E ek sich ände nde Umwel ak o en sowie die Rele anz wei e e LMWOS-Senken
(So p ion und P lanzenau nahme) wu den anhand meh e e zusä zliche Labo expe imen e
mi posi ionsspezi ische
14
C Ma kie ung e aluie . Die Ein üh ung des Di e genz Index (DI)
e möglich e es den posi ionsspezi ischen Einbau in e schiedenen S udien unabhängig om
applizie en Iso op, dem expe imen ellen Design und dem un e such en Pool zu e gleichen.
Mik oo ganismen wa en die dominan e Senke ü LMWOS und wede P lanzenau -
nahme noch So p ion konn en in Ra e und Kine ik mi mik obiellen Au nahmesys emen kon-
ku ie en. De Einbau einzelne Molekülposi ionen in Boden, mik obielle Biomasse und be-
s imm e Subs anzklassen wa du ch den mik obiellen Me abolismus bes imm , .a. du ch
Glykolyse, Oxida ion du ch Py u a -Dehyd ogenase und Ci a zyklus. Alle dings lie en pa al-
lel zu diesen oxidie enden, ka abolen S o wechselwegen auch anabole Reak ionen, d. h. de
Au bau neue Zellkomponen en, ab. Dies üh e zu einem s a ken C-Umsa z und Recycling,
nich nu im Cy osol sonde n z.B. auch on Zellwandpolyme en. In ensi e Umsä ze inne halb
me abole Sei enäs e, wie de Fe säu ebiosyn hese, wu den iden i izie . Diese E gebnisse
zu Fe säu e ans o ma ion sind wesen lich ü die Anwendung on Fe säu en als p lanzli-
che Bioma ke in Paläoumwel s udien.
Die Kombina ion posi ionsspezi ische
13
C Ma kie ung mi komponen enspezi ische
Iso openanaly ik mik obielle Bioma ke e laub e des Wei e en die Iden i ika ion spezi ische
S o wechselwege einzelne mik obielle G uppen. Pilze zeig en einen langsame en in azel-
Zusammen assung
XXI
lulä en C-Umsa z als Bak e ien, was die me abole G undlage ü den langsam-zyklie enden,
pilzbasie en und den schnell-zyklie enden, bak e ienbasie en Zweig des Bodennah ungs-
ne zes lie e . Die Ve schiebungen de Kohlens o lüsse du ch e schiedene S o wechsel-
wege wu den in Abhängigkei on Umwel ak o en iden i izie : Mi Zunahme de Subs a -
konzen a ion konn e ein G adien on C-Mangel-S o wechselwegen übe den E hal ungs-
me abolismus hin zu cha ak e is ischen Wachs ums-S o wechselwegen beobach e we den.
Eine Ve inge ung de Subs a e ügba kei du ch So p ion e u sach e eine ähnliche Ve -
schiebung de me abolen C-Flüsse: Je nied ige die Ve ügba kei , des o meh C wi d in Bio-
syn hesewege also mik obielle P oduk e, e lage .
Diese S udien konn en zeigen, dass posi ionsspezi ische Ma kie ung nich nu eine
we olle Me hode in de Biochemie da s ell , sonde n auch die Au klä ung de Ve s o -
wechslung on LMWOS du ch di e se mik obielle Gemeinscha en in komplexen Medien wie
dem Boden e möglich . Pa allel ablau ende P ozesse in Böden wie z. B. 1) de Rück luss
du ch e e sible S o wechselwege, 2) Umsä ze in e schiedenen mik obiellen G uppen ode
3) Umsä ze in spezi ischen Mik ohabi a en (an Mine alobe lächen, am Boden-P lanze-
In e ace ode an Ho -spo s e sus dem Gesam boden) können mi els posi ionspezi ische
Ma kie ung im Boden in si u e olg we den.
De Umsa z on LMWOS in Ka a- und Anabolismus wu de im Rahmen diese Disse a-
ion ekons uie . Das Ve s ändnis ü diese S o wechselwege und ih e Regula ions ak o en
is en scheidend ü die Beu eilung on C-Flüssen zwischen Mine alisa ion und dem Au bau
mik obielle Biomasse – de Vo ausse zung zu Bildung mik obielle , o ganische Bodensub-
s anz. Das Wissen übe T ans o ma ionssch i e und ih e egulie enden Fak o en is essen-
iell ü die Vo he sage (z. B. mi els p ozessbasie e Modellie ung), abe auch ü die Mani-
pula ion de C-Seques ie ung und S abilisie ung in Böden.
Zusammen assung
XXII
Ex ended Summa y
1
1 Ex ended Summa y
1.1 In oduc ion
1.1.1 Low molecula weigh o ganic subs ances in soils
1.1.1.1 Role and ele ance o low molecula weigh o ganic subs ances in soil
Soil o ganic ca bon (SOC) is he la ges e es ial ca bon (C) pool, wi h C s ocks o
a ound 1500 Pg (Ba jes, 1996). On a e age 30-120 kg C m
-2
is s o ed up o 1 m soil
dep h and 0.5 o 1% o his s ock is annually espi ed ( an Hees e al., 2005a) bu he
la ges po ion is s able o inac i e. T adi ionally, SOC has been di ided ope a ionally by
chemical ac iona ion in o s uc u ally di e se ul ic and humic acids ( an Hees e al.,
2005a). Mo e ecen esul s e ealed ha a limi ed ange o de ined subs ance classes
and hei polyme s build up he soil o ganic ma e (SOM) (Schmid e al., 2011; on
Lue zow e al., 2006).
A mino po ion o SOC cons i u es dissol ed o ganic ma e (DOC), in mos cases
less han 2 mol C m
-2
( an Hees e al., 2005a). Only up o 10% o DOC consis s o iden i-
iable compounds o low molecula weigh . These low molecula weigh o ganic sub-
s ances (LMWOS) a e de ined as soluble subs ances wi h a molecula weigh lowe han
250 Da (Boddy e al., 2007) and mainly consis o alipha ic and a oma ic ca boxylic acids,
amino acids and pep ides, mono-, di- and small oligosaccha ides, amino suga s, phenolic
subs ances and side opho es (McKeague e al., 1986).
Al hough he po ion o LMWOS in SOC is ex emely low, hey play a majo ole in
ecosys em unc ions. Rega ding he C cycle, he impo ance o LMWOS is no de e -
mined by hei pool size (Fische e al., 2007), bu by hei huge luxes (>20 mol C m
-2
y
-1
)
ha pass h ough his pool. Du ing decomposi ion o plan -de i ed o ganic ma e he
high molecula weigh o ganic subs ances a e deg aded by exoenzymes in o low molecu-
la monome s and pass he pool o LMWOS. They can hen be oxidized o CO
2
by mic o-
bial espi a ion. Van Hees e al (2005a) summa ized o o es soils ha al hough
LMWOS comp ise less han 0.05% o he C pool, hey con ibu e o mo e han 10-20% o
he soil espi a o y luxes, hus demons a ing he high ele ance o his ac i e, as cy-
cling C pool o he SOC u no e .
In addi ion o hei unc ion as ene gy and C sou ce o mic oo ganisms, hey ul ill
se e al impo an unc ions in soils: 1) Con ibu ion o wea he ing and solubiliza ion o
nu ien s o plan s; 2) o ma ion o soil s uc u es like agg ega es; 3) accele a ion o e-
Ex ended Summa y
8
1.1.4 Objec i es
The main objec i e o his hesis was o es ablish posi ion-speci ic
13
C- and
14
C-
labeling and me abolic acing as a ool in soil science, which enables a p ocess-
o ien a ed iew on he LMWOS cycle and can be applied o ield and labo a o y expe i-
men s. Mo e speci ically, he ollowing objec i es we e aimed owa ds:
1) De e mina ion o me abolic pa hways o wo ep esen a i es o he h ee main
subs ance classes o LMWOS (amino acids, monosaccha ides and o ganic
acids) by posi ion-speci ic labeling
2) Coupling o posi ion-speci ic labeling wi h compound-speci ic iso ope analysis
o mic obial bioma ke s
a. o ollow he C inco po a ion in o a ious cellula compound classes
b. o iden i y speci ic me abolic pa hways o indi idual membe s o he
soil mic obial communi y
3) Iden i ica ion o speci ic me abolic pa hways depending on ce ain en i on-
men al condi ions:
a. pa hways o LMWOS so bed on a ious soil componen s
b. pa hways unde a ious concen a ions o LMWOS
c. ex a- e sus in acellula ans o ma ion pa hways
4) Assessmen o kine ics and ecological ele ance o compe ing sinks o
LMWOS:
a. so p ion e sus mic obial u iliza ion
b. plan up ake e sus mic obial u iliza ion
1.2 Expe imen s and Me hods
1.2.1 Field expe imen
The ield expe imen was ca ied ou on an ag icul u al ield si e close o Hohen-
poelz (49°54' No he n la i ude; 11°08' Eas e n longi ude, 500 m a.s.l.) in no he n Ba a-
ia. Mean annual empe a u e is +7 °C, mean p ecipi a ion is 870 mm and soil ype is a
haplic Lu isol (IUSS Wo king g oup WRB, 2007). 784 columns (Figu e S2) we e ins alled
acco ding o a andomized block design, whe e he ou blocks ep esen he ou eplica-
ions o each ea men .

Ex ended Summa y
9
Fig. S2 Schema ic (le ) and pho o (middle) o a labeling column o he ield expe i-
men . Righ pho o shows one o he ou blocks, each wi h 196 columns.
In o al, six indi idual LMWOS we e applied: wo amino acids (alanine and glu a-
ma e), wo monosaccha ides (glucose and ibose) and wo o ganic acids (applied as acid
anion: ace a e and palmi a e). Each o hei pu chasable iso opome s as well as hei
uni o mly
13
C-labeled o m was applied in sepa a e columns (see Table S1). In addi ion,
15
N-labeled glu ama e and alanine we e added as well as labeled mine al ni ogen
(
15
NO
3
- and
15
NH
4+
). Consequen ly, his is he i s expe imen which allows me abolic
acing by wo pa allel app oaches: 1) me abolic acing based on indi idual C posi ions
(Dijks a e al., 2011a) and 2) me abolic acing based on mul iple iso ope labeling
(Knowles e al., 2010) ( esul s o he second app oach we e no included in his hesis).
Each o he six subs ances had one backg ound ea men whe e he same amoun
o non
13
C-en iched subs ance was applied. In each o he 22 labeled and 6 backg ound
ea men s, he amoun o C and N applied was as low as possible and iden ical o all
columns o a oid po en ial dis u bance o he mic obial communi y. Each ea men was
designed o se en sampling da es and in ou eplica ions ( esul ing in 784 soil columns).
To p e en ain all and hus leaching, a oo was ins alled abo e he ield si e du ing
labeling and o he i s 10 days. Excluding addi ional ain all wa e due o he oo and
assuming no p e e en ial low in he eshly illed soil, leaching can be ega ded as negli-
gible and mine aliza ion is he only p ocess emo ing
13
C om he soil. Only da a om
he i s wo sampling da es, e lec ing he sho - e m dynamics o LMWOS, a e p e-
sen ed in his hesis.
Sampling was pe o med by emo ing he en i e column om he ield. Leng h (i.e.
olume) o soil column, esh weigh o soil and wa e con en we e de e mined. A e
homogeniza ion, each sample was spli and he subsamples we e p epa ed and s o ed
acco ding o he di e en analyses.
10 cm
13 cm 10 cm
10 cm
13 cm 10 cm
Ex ended Summa y
10
Table S1 T ea men s o posi ion-speci ic
13
C and
15
N labeling. Applied amoun o
13
C and
15
N, hei iso opic en ichmen , as well as he espec i e compound and labeled
posi ion a e p esen ed. Na . abund. means applica ion o non-en iched sub-
s ances, x means“no applica ion o
15
N in his ea men .
Posi ion-speci ic
13
C labeling
15
N labeling
subs ance
class subs ance posi ion(s)
applied amoun
o
13
C (µmol
pe column) subs ance
applied amoun
o
15
N (µmol
pe column)
C-1 94.2 x
C-2 93.0 x
C-3 94.7
alanine
(na . abund.) x
alanine
uni o mly 96.3
15
N alanine
(98 a %) 28.7
C-1 93.0 x
C-2 56.3 glu ama e
(na . abund.) x
amino
acids
glu ama e
uni o mly 91.6
15
N glu ama e
(98 a %) 28.0
C-1 91.5 x
C-2 93.4 x
C-4 93.5 x
C-6 91.8
(NH
4
)
2
SO
4
(na . abund.)
x
glucose
uni o mly 93.4
15
N (NH
4
)
2
SO
4
(98 a %) 27.8
C-1 93.3 x
C-5 93.0 x
mono-
sacha ides
ibose
uni o mly 91.8
(NH
4
)
2
SO
4
(na . abund.) x
C-1 94.6 x
C-2 94.1 x
ace a e
uni o mly 95.8
KNO
3
(na . abund.) x
C-1 47.5 x
C-2 47.1 x
C-16 44.3
KNO
3
(na . abund.) x
o ganic
acids palmi a e
uni o mly 49.5
15
N KNO
3
(98 a %) 28.6
1.2.2 Labo a o y expe imen s
Besides he ield expe imen , se e al labo a o y expe imen s wi h modi ica ions o
en i onmen al condi ions we e pe o med. In gene al, in labo a o y expe imen s posi ion-
speci ic
14
C-labeled ace s we e applied.
1.2.2.1 Expe imen 1: T ans o ma ions o ee alanine
The i s expe imen aimed a iden i ying he ans o ma ion pa hways o amino
acids depending on wo ac o s: 1) he concen a ion o alanine (0.5, 5, 50, 500 and
5000 µM), and 2) he ex a- and in acellula as well as abio ic p ocesses (i.e. so p ion) o
alanine emo al om soil solu ion, sepa a ed by selec i e inhibi ion. Alanine
ans o ma ion p oduc s we e ope a ionally sepa a ed by sequen ial ex ac ion in o ion-
exchangeable and ligand-exchangeable ans o ma ion p oduc s, whe eas i e e sible-
bound ans o ma ion p oduc s emained in he soil.
Ex ended Summa y
11
1.2.2.2 Expe imen 2: T ans o ma ions o so bed alanine
In he so p ion expe imen , a ailabili y and mic obial u iliza ion pa hways o ab-
so bed alanine we e in es iga ed in wo s eps:
1) Labeled alanine was adso bed o i e s e ilized so ben s commonly p esen in
soils: wo i on oxides wi h di e en c ys alline s uc u e: goe hi e and hema i e; wo clay
mine als wi h 2:1 laye s – smec i e, and 1:1 laye s – kaolini e; and ac i e coal.
2) The ea e , he so bed alanine was mixed wi h he soil and incuba ed o 3 days.
The e ec o so p ion on mic obial u iliza ion, especially hei me abolic pa hways, was
elucida ed.
1.2.2.3 Expe imen 3: Plan up ake o in ac alanine
Plan up ake was ca ied ou wi h dual-iso ope, posi ion-speci ic labeled alanine in
hizo ubes (Bie na h e al., 2008; Rasmussen e al., 2010). Up ake o C and N om indi-
idual posi ions by Zea mays, Lupinus albus and Cicho ium in ybus was aced. As a
con ol,
14
C ace a e and mine al
15
NH
4+
and
15
NO
3-
we e applied. Thus, passi e up ake o
LMWOS as well as he ele ance o N-LMWOS up ake compa ed o mine al ni ogen was
assessed. Posi ion-speci ic labeling enabled he di e en ia ion o in ac alanine up ake
e sus he up ake o i s ans o ma ion agmen s.
1.2.3 Me hods o ace
13
C and
14
C in ans o ma ion p od-
uc s o LMWOS
An o e iew o e he me hods, applied in his hesis, o ace
13
C,
15
N and
14
C in
speci ic ans o ma ion p oduc s and mo e unspeci ic SOC ac ions is p esen ed in Table
S2. Me hods a e b ie ly desc ibed in he ollowing chap e s and in de ail in he Ma e ial
and Me hods sec ion o he espec i e s udies.
Ex ended Summa y
12
Table S2 Applied
13
C- and
14
C-labeling app oaches as well as analy ical me hods o he
indi idual s udies; Fi s line shows whe he samples we e de i ed om ield o
labo a o y expe imen s; PS indica es posi ion-speci ic labeling.
Field expe imen Labo a o y expe imen
S udy S udy 1 S udy 2 S udy 3 S udy 6 + 7 S udy 8 S udy 4 S udy 5 S udy 9
Topic LMWOS
com-
pa ison
me hod
de elop-
men
amino
acid
mono-
saccha-
ides
o ganic
acids
ee
amino
acids
so bed
amino
acids
plan up-
ake o
amino
acids
Labeling Uni o mly
13
C PS
13
C PS
13
C PS
13
C PS
14
C PS
14
C PS
14
C
&
15
N
13
C in
bulk soil
X X X X
15
N in
bulk soil
X
15
N in plan
biomass
X
mic obial
biomass
13
C
X X X X
13
C in
PLFA
X X X X
13
C in amino
suga s
X X
14
C in soil
solu ion
X X
14
C in
bulk soil
X X X
14
C in soil
ex ac s
X
14
C in
CO
2
X
1.2.3.1 Bulk-iso ope measu emen s by EA-IRMS
13
C and
15
N emaining in soil was quan i ied by de e mina ion o δ
13
C and δ
15
N al-
ues o bulk soil samples. Inco po a ion o
13
C in mic obial biomass was calcula ed om
he δ
13
C alue o umiga ed and un umiga ed soil ex ac s, gained by he chlo o o m-
umiga ion-ex ac ion me hod (B ookes e al., 1985; Wu e al., 1990). Fo all δ
13
C meas-
u emen s, he samples we e eeze-d ied and measu ed by Elemen al Analyze -Iso ope
Ra io Mass Spec ome e (EA-IRMS).
13
C inco po a ion was calcula ed acco ding o he
mixing model e e enced on he espec i e δ
13
C alues o he backg ound ea men .
1.2.3.2 Compound-speci ic iso ope analysis o mic obial bioma ke s
13
C inco po a ion in o mic obial memb ane lipids, he phospholipids a y acids
(PLFA), was de e mined by compound-speci ic iso ope analysis. Fo his, he Bligh-and-
Dye ex ac o in ac pola lipids was pe o med. PLFA we e pu i ied by liquid-liquid ex-
ac ion and column ch oma og aphy and de i a ized o hei a y acid me hyl es e s
(FAMEs). δ
13
C alue o FAMEs was de e mined by gas ch oma og aphy-combus ion-
Ex ended Summa y
13
iso ope a io mass spec ome y (GC-C-IRMS) and
13
C inco po a ion was calcula ed by
mixing models wi h he espec i e backg ound ea men s as a e e ence.
In addi ion,
13
C inco po a ion in o mic obial cell wall monome s, he amino suga s,
was measu ed in his hesis. Howe e , GC-C-IRMS me hods a e no su icien ly eliable
and exis ing liquid ch oma og aphy-oxida ion-iso ope a io mass spec ome y me hods
we e no able o de e mine he δ
13
C alue o bac e ial mu amic acid. The e o e, a new
ins umen coupling, using an ion ch oma og aph (IC) ins ead o a classical liquid ch o-
ma og aph o IC-O-IRMS measu emen s and amino suga δ
13
C de e mina ion was es-
ablished in s udy 2 (see Figu e S3). Thus, p e ious pu i ica ion me hods (Bode e al.,
2009; Glase and G oss, 2005; Indo e al., in p ess) had o be op imized and an IC-O-
IRMS measu emen had o be elabo a ed (see s udy 2). B ie ly, a e acid hyd olysis, i on
and sal s we e emo ed by p ecipi a ion (Zhang and Amelung, 1996) and neu al com-
pounds we e sepa a ed om amino suga s by a ca ion exchange esin (Indo e al.,
2013). Liquid ch oma og aphy was op imized (Bode e al., 2009) and
13
C inco po a ion
in o mic obial cell walls was calcula ed using he analogy o inco po a ion in o PLFA.
Fig. S3 O e iew o he ins umen coupling: Ion Ch oma og aph is shown on he le
side wi h he pump, au osample and de ec o -ch oma og aphy compa men .
Connec ion o isolink occu s ia a PEEK capilla y wi h in e posed colloid il e .
Scheme o LC Isolink is adap ed om K ummen e al. (2004).
1.2.3.3 Radiochemical analyses
Inco po a ion o
14
C in o he ans o ma ion p oduc s was pe o med a e a ious
sampling and ex ac ion me hods based on scin illa ion coun ing. Fo his pu pose, solid
samples we e combus ed and
14
CO
2
was apped in NaOH. Bound
14
C was de e mined
by scin illa ion coun ing.
14
C in soil ex ac s o suspensions could be di ec ly measu ed

Ex ended Summa y
14
a e mixing a subsample wi h a scin illa ion cock ail. Di ec measu emen o decomposed
14
CO
2
was pe o med on expe imen s in well pla es. The e o e, a CO
2
ap based on 24-
segmen il e pape was cons uc ed abo e he 24-well pla e. Cons uc ion o his ap
was op imized and e iciency e alua ed in s udy 5.
1.2.4 The Di e gence Index
I emains challenging o compa e he posi ion-speci ic a e o indi idual LMWOS,
especially i C is ans o med in o s ongly di e ing C pools. To enable he compa ison o
he indi idual s udies independen o he iso opic app oach (
13
C o
14
C) o expe imen al
design used o he pool in es iga ed, he Di e gence Index DI
i
was in oduced in his he-
sis (equa ion 1).
[
]
[ ]
∑
=
=
⋅
=
ni
1i
i
i
i
C
Cn
DI (1)
This index shows he a e o indi idual C a oms om he posi ion i wi hin a ans-
o ma ion p ocess ela i e o he mean ans o ma ion o he n o al numbe o C a oms in
he subs ance. Thus, a DI
i
o 1 means ha he ans o ma ion o his C a om in he in es-
iga ed pool co esponds o ha o uni o mly labeled subs ance (a e age o all C a oms o
he subs ance). The DI
i
anges om 0 o n, and alues be ween 0 and 1 e lec educed
inco po a ion o he C in o he in es iga ed pool, whe eas alues be ween 1 and n show
inc eased inco po a ion o he C a om in o his pool as compa ed o he a e age. This
index is no dependen on absolu e amoun s o p opo ions o he subs ance used in indi-
idual p ocesses. The e o e, i enables compa ison o he dis ibu ion o indi idual C a -
oms o e he whole ange o in es iga ed concen a ions, he size o C pools, he p ocess
a es, e c.
Ex ended Summa y
15
1.3 Resul s and Discussion
1.3.1 O e iew: main esul s o he s udies
An o e iew o e he objec i es and he main esul s and conclusions o he indi-
idual s udies is p esen ed in Table S3.
Table S3 Ti le o he indi idual s udies as well as hei objec i es and main conclusions.
S udy Objec i es Main Conclusions
S udy 1:
Fa e o low molecu-
la weigh o ganic
subs ances in soil:
om mic obial up-
ake o u iliza ion
and s abilisa ion
• O e iew o mic obial me abolism
and C alloca ion wi hin he mic obial
me abolism
• Iden i ica ion o speci ics in he
LMWOS u iliza ion o a ious mi-
c obial g oups in soil
• The en y s eps o an
LMWOS o he basic C me-
abolism accoun s o he
a e o he 3 classes o
LMOWS in soil
S udy 2:
Imp o ed δ
13
C
analysis o amino
suga s in soil by Ion
Ch oma og aphy -
Oxida ion - Iso ope
Ra io Mass Spec-
ome y
• Es ablishmen o a new ins umen
coupling o ion ch oma og aph wi h
iso ope a io mass spec ome e s
o ou ine measu emen s o wa e -
dissol able me aboli es by IRMS
• De elopmen o an IC-O-IRMS
me hod o pa allel amino suga
quan i ica ion and δ
13
C de e mina-
ion
• IC-O-IRMS enables eliable
and ou ine measu emen s
o amino suga s
• Pa allel quan i ica ion and
δ
13
C de e mina ion o basic
and acidic amino suga s is
possible in soils
• IC-O-IRMS has g ea ad an-
ages compa ed o classical
LC-O-IRMS
S udy 3:
Biochemical pa h-
ways o amino acids
in soil: Assessmen
by posi ion-speci ic
labeling and
13
C-
PLFA analysis
• Iden i ica ion o ans o ma ion
pa hways o wo ep esen a i e
amino acids (alanine and glu a-
ma e)
• Assessmen o speci ics in amino
acid me abolism o indi idual mi-
c obial g oups in soils
• Basic C me abolism ac-
coun s o he majo i y o he
obse ed alanine ans o -
ma ions
• Inco po a ion o glu ama e
C-2 e lec s speci ic mic o-
bial pa hways
S udy 4:
Biogeochemical
ans o ma ions o
amino acids in soil
assessed by posi-
ion-speci ic labeling
• E ec o concen a ion (i.e. C a ail-
abili y) on alanine ans o ma ions
• Kine ics o so p ion, ex acellula
ans o ma ion and mic obial up ake
and u iliza ion o alanine
• Bio ic p ocesses ou compe e
so p ion in soils
• Ex acellula LMWOS ans-
o ma ions a e only ele an
a low concen a ions o in
special mic ohabi a s
• Concen a ion s ongly a -
ec s he me abolic pa hway
and a e o alanine C in soils
S udy 5:
So p ion a ec s
amino acid pa hways
in soils: Implica ion
om posi ion-
speci ic labeling o
alanine
• Mic obial a ailabili y o LMWOS
so bed o a ious so ben s
• E ec o so p ion on mic obial u ili-
za ion and ans o ma ion pa hway
o LMWOS
• T ans o ma ion o so bed
LMWOS ollows classical
biochemical pa hways: no
abio ic ans o ma ions occu
• C alloca ion h ough speci ic
pa hways in mic obial me-
abolism is s ongly a ec by
he so p ion mechanism and
bioa ailabili y o LMWOS
Ex ended Summa y
16
S udy 6:
Biochemis y o hex-
ose and pen ose
ans o ma ion in soil
analyzed by posi-
ion-speci ic labeling
and
13
C-PLFA
• T acing me abolism pa hways o
hexoses and pen oses in soils
• Iden i ica ion o speci ics in bac e ial
and ungal me abolism in he use o
monosaccha ide C o PLFA o ma-
ion
• Glycolysis and pen ose
phospha e pa hway as well
as back lux could be aced
in soils in si u
• Glucose – as a ubiqui ous
subs a e – is sp ead o e
en i e me abolism, in en-
si ely ecycled and a la ge
po ion is used o mic obial
biomass o ma ion
S udy 7:
Me abolic pa hways
o ungal and bac e-
ial amino suga
o ma ion in soil
assessed by posi-
ion-speci ic
13
C-
labeling
• Recons uc ion o he main pa h-
ways o amino suga biosyn hesis
in soils
• Iden i ica ion o speci ics in bac e ial
and ungal me abolism du ing cell
wall o ma ion
• Fo ma ion o amino suga s
ollows se e al pa hways: 1)
di ec in ac glucose u ilia-
ion, 2) glycolysis and back-
lux and 3) pen ose phos-
pha e pa hway and back lux
• Fungi showed a lowe me a-
bolic ac i i y han bac e ia in
main enance me abolism
S udy 8:
Fo ma ion and ans-
o ma ion o a y
acids in soils as-
sessed by posi ion-
speci ic labeling o
p ecu so s
• Recons uc ion o he main pa h-
ways o a y acid o ma ion and
ans o ma ion in soils
• Iden i ica ion o mic obial g oup
speci ic a y acid me abolism
• Assessmen o mic obial ans o -
ma ion o he ee a y acid pool in
soils
• Ace a e
13
C was a ely used
o building o a y acid
backbones bu mainly o
elonga ions o in oduc ions
o unc ional g oups
• Fa y acids we e in ensi ely
modi ied in soils acco ding o
he demand o he mic obial
communi y which has o be
conside ed o paleoen i-
onmen al applica ions
S udy 9:
O ganic N up ake by
plan s - Ree alu-
a ion by posi ion-
speci ic labeling o
amino acids
• De e mina ion o he o e es ima ion
o uni o mly labeling app oaches
• E alua ion o he ele ance o in ac
amino acid up ake o he N nu i-
ion o ag icul u al plan s
• Quan i ica ion o in ac
amino acid up ake based on
uni o m labeling causes an
1.2-3 old o e es ima ion
• Mic obial u iliza ion s ongly
domina ed he a e o o -
ganic subs ances in soils
• The majo i y o amino acid
up ake by plan s was ex-
plained by passi e up ake o
mic obial ans o ma ion
p oduc s
1.3.2 De e mina ion o me abolic pa hways o amino acids,
monosaccha ides and o ganic acids
1.3.2.1 Simila i ies and di e ences o indi idual LMWOS
S udy 1 aimed a in es iga ing he simila i ies and di e ences o he a e o he ap-
plied LMWOS, o gain insigh s in hei mic obial u iliza ion and decomposi ion. The pe -
cen age o
13
C-LMWOS emaining in he SOM and inco po a ed in o he mic obial bio-
mass pool a e 3 days was simila o he subs ances in es iga ed (Figu e S4). Howe e ,
a e 10 days, signi ican di e ences be ween he inco po a ion o indi idual subs ances
Ex ended Summa y
17
a ose. The ini ial apid up ake was qui e simila o all in es iga ed LMWOS as hey a e
ubiqui ous subs a es o many membe s o he mic obial communi y. Howe e , in acel-
lula me abolism may ha e accoun ed o he obse ed di e ences in he a e o
LMWOS-C in soil and his e ec became e iden a e 10 days o con inued ans o ma-
ion o he LMWOS (Figu e S4).
Fig. S4
13
C eco e y (in % o applied
13
C) om six LMWOS in soil, mic obial biomass
and PLFA, 3 and 10 days a e addi ion. Le e s indica e signi ican di e ences
in
13
C inco po a ion o he indi idual subs ances i occu ing.
LMWOS speci ics o inco po a ion in o he mic obial biomass depend on hei en y
poin in o he basic C me abolism o mic oo ganisms (Figu e S5): Mic obial up ake o
bo h amino acids was simila on day 3, bu much less glu ama e C han alanine C e-
mained in mic obial biomass on day 10 (Figu e S4). This e lec s he ac ha subs a es
wi h di ec inco po a ion in o he oxidizing ci ic acid cycle (Figu e S5), such as glu ama e,
a e p e e en ially oxidized o ene gy p oduc ion compa ed o LMWOS, like alanine,
which en e glycolysis. The high and apid glucose up ake is connec ed o he ac ha
glucose is he mos abundan suga in soils (De ien e al., 2006; De ien e al., 2004;
Fische e al., 2010a). In compa ison o amino acids and ca boxylic acids, glucose was
p e e en ially inco po a ed in o mic obial biomass. This e lec s he p e e ence o glycoly-
sis subs a es o anabolic u iliza ion compa ed o ca abolism. Fo ca boxylic acids, he
13
C inco po a ed in mic obial biomass declined by a ac o o wo om day 3 o day 10,
which also e lec s he p e e ed ca abolic oxida ion o subs ances en e ing he ci ic acid
cycle (Figu e S5).
Inco po a ion o LMWOS
13
C in o mic obial memb ane lipids, he PLFA, enables he
iden i ica ion o p e e ences o indi idual unc ional mic obial g oups o ce ain subs a es
Ex ended Summa y
24
o plan -de i ed a y acid inge p in s and hei iso ope signa u e o paleoen i onmen al
s udies is hampe ed.
Fig. S7 Me abolic pa hways o a y acid o ma ion om ace a e and a y acid ans o -
ma ions o palmi a e in soil.
1.3.3 Iden i ica ion o speci ic me abolic pa hways
1.3.3.1 Speci ic pa hways o indi idual membe s o he mic obial communi y
in soils
The in es iga ed compound classes o mic obial p oduc s a e on he one hand spe-
ci ic o a ce ain biosyn he ic pa hway wi hin mic obial cells. On he o he hand, PLFA
and he amino suga s a e also bioma ke s o indi idual mic obial g oups in soils. This
enables he iden i ica ion o speci ic pa hways o indi idual membe s o he soil mic obial
communi y.
Amino suga s enable ungi and bac e ia o be dis inguished be ween (Engelking e
al., 2007, Glase e al., 2004): In s udy 7, bac e ial mu amic acid showed he highe dy-
namics o
13
C eplacemen o he cell walls (s udy 7, Figu e 3). In addi ion he DI o indi-
idual C posi ions changed s ongly om day 3 o day 10 o bac e ial mu amic acid bu
emained cons an o ungal galac osamine (s udy 7, Figu e 2). This e lec s a mo e ac-
i e main enance me abolism in bac e ia, which caused an inc easing inco po a ion o
glucose me aboliza ion agmen s wi hin a pe iod o 10 days. The e o e, his s udy p o ed
o he i s ime ha di e ences in he C u no e in he slow- and as -cycling b anch o
soil ood webs can be a ibu ed o me abolic p ocesses (Moo e e al., 2005).
A mo e de ailed inge p in o he mic obial communi y, especially o he p oka y-
o ic g oups, can be gained by indi idual PLFA (Moo eKuce a and Dick, 2008). Combining
s a is ical g ouping o he a y acids wi h known a y acid inge p in s enables unc ional

Ex ended Summa y
25
mic obial g oups o be dis inguished in soil (s udies 3, 6 and 8). The DI e ealed o glu-
ama e inco po a ion in o PLFA ha wo unc ional mic obial g oups – g am-posi i e II
and ungi – showed a speci ic C alloca ion: Glu ama e C-2 was ans e ed om he ci ic
acid cycle owa ds a y acid syn hesis. Special eac ions (e.g. he glyoxyla e bypass) a e
necessa y o a oid he oxida ion o glu ama e C-2 (Caspi e al., 2012). None o he o he
mic obial g oups showed C-2 inco po a ion in o PLFA (see s udy 3, Figu e 4). Conse-
quen ly, his was he i s ime ha speci ic pa hways o indi idual membe s o he soil
mic obial communi y could be aced in soils in pa allel.
1.3.3.2 Pa hways unde a ious concen a ions o LMWOS
C alloca ion wi hin me abolic pa hways is s ongly a ec ed by en i onmen al condi-
ions. The bioa ailabili y o C sou ces, especially LMWOS, is one o he main ac o s con-
olling he s a e o he mic obial communi y - om main enance o g ow h
(Blagoda skaya e al., 2007, Fische e al., 2010b; Schneckenbe ge e al., 2008). In
s udy 4, alanine ans o ma ions we e in es iga ed wi hin an alanine concen a ion g adi-
en ep esen ing he ull ange o concen a ions in soil: om ba e soil (0.5 µM) o ho
spo s (5 mM).
A low alanine concen a ion, up ake was desc ibed by Michaelis-Men en kine ics,
whe eas wi h inc easing concen a ion, linea kine ics domina ed mic obial up ake (Jones
and Hodge, 1999). This shi in up ake kine ics e ealed ha wo mechanisms we e e-
sponsible o he mic obial up ake: An unsa u able, unspeci ic up ake o in ac alanine a
ho -spo concen a ions and speci ic, ac i e up ake mechanisms a low alanine concen-
a ions. The DI o he non-ex ac able pool o mic obial ans o ma ion p oduc s (con ain-
ing mac omolecules as well as lipids) e lec ed addi ional shi s in he in ensi y o he
alanine me abolism pa hways (Figu e S8).
A signi ican ly inc eased inco po a ion o C-1 unde lowes C a ailabili y may indi-
ca e C s a a ion pa hways e.g. anapleu o ic pa hways. A con e gence o he DI o C-2
and C-3 o highes alanine concen a ion is cha ac e is ic o many anabolic pa hways o
biomass o ma ion unde g ow h condi ions (e.g. lipid biosyn hesis om alanine o he
o ma ion o new cell memb anes: see igu es S5 and S7).
Ex ended Summa y
26
Fig. S8 Concen a ion-dependen posi ion-speci ic ans o ma ion index DI
i
(N=6, ±
SEM) o alanine C posi ion inco po a ed in o he no -ex ac able pool o mic o-
bial biomass compounds.
These and u he esul s om s udy 4 indica e an al e ed C alloca ion in o indi id-
ual pa hways ha is dependen on subs a e a ailabili y: om anabolic pa hways cha ac-
e is ic o C de iciency ia main enance me abolism owa ds pa hways common o
g owing cells (Figu e S9).
Fig. S9 Gene al biochemical pa hways o amino acids me aboliza ion in soil as depend-
ing on alanine a ailabili y. Line wid h ep esen s he quali a i ely es ima ed ela-
i e shi s o alanine C be ween ce ain pa hways dependen on he alanine
concen a ion.
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.5 µM 5 µM 50 µM 500 µM 5000 µM
DI
i
C-1 Alanine
C-2 Alanine
C-3 Alanine
Ex ended Summa y
27
1.3.3.3 Pa hways o so bed LMWOS
So p ion is one o he mos likely p ocesses causing he long- e m s abiliza ion o C
in soils (Gonod e al., 2006, Duemig e al., 2012). Ne e heless, a pa o he so bed
LMWOS emains bioa ailable: S udy 5 showed o alanine ha a leas 20-50% o he
mine al-so bed alanine was mic obially me abolized. Howe e , so p ion educed he
a ailabili y and consequen ly a ec ed ans o ma ion pa hways (Jones and Edwa ds,
1998). In s udy 5, ans o ma ions o posi ion-speci ically labeled alanine, so bed o i e
so ben s ( wo i on oxides wi h di e en c ys alline s uc u e: goe hi e and hema i e; wo
clay mine als wi h 2:1 laye s – smec i e, and 1:1 laye s – kaolini e; and ac i e coal) we e
in es iga ed. Goe hi e and ac i e coal showed he highes amoun o so bed alanine
(~45% o added alanine), and he lowes po ion o he so bed alanine C was mic obially
u ilized (26 and 22%, espec i ely), whe eas clay mine als showed lowe so p ion (10-
26% o added alanine) and a highe po ion ha was mic obially a ailable (30-35%).
The s onge he so p ion by he indi idual so ben , he lowe he mic obial u iliza-
ion was (Jones and Hodge, 1999). The a e o indi idual molecule posi ions e lec ed
ha , a leas o he ou mine al phases, alanine was p ocessed by he classical bio-
chemical pa hways: deamina ion, deca boxyla ion o C-1 by py u a e dehyd ogenase and
u he oxida ion o C-2 and C-3 in he ci ic acid cycle (Djiks a e al., 2011). Howe e ,
he in ensi y o mic obial pa hways depended on he bioa ailabili y o he so bed sub-
s a e: he less alanine was accessible, he less was oxidized by ca abolism and he mo e
alanine C was used o anabolism, i.e. he o ma ion o mic obial biomass.
0
2
4
6
8
0 10 20 30 40 50 60 70 80
ime (h)
a io C-1/C-2,3 in espi ed CO2
heama i e
goe hi e
smec i e
kaolini e
ac i e coal
Fig. S10 Ra io o C-1 o (C-2+C-3)/2 espi a ion o alanine C o he 5 applied so ben s
calcula ed om he i ed, posi ion-speci ic oxida ion a e.
Ex ended Summa y
28
The a io o C-1 oxida ion by py u a e dehyd ogenase e sus oxida ion o C-2 and
C-3 in he ci ic-acid cycle (Figu e S10) depended on he mic obial a ailabili y o alanine:
High a ailabili y due o as deso p ion o ca ion-exchangeable bound alanine caused an
ini ial peak in C-1 oxida ion by glycolysis o he wo clay mine als and an ab up shi o
oxida ion ia he ci ic acid cycle. Howe e , low mic obial a ailabili y o alanine so bed o
i on mine als led o a pa allel oxida ion o all h ee posi ions by glycolysis and ci ic acid
cycle ( ep esen ed by he C-1/C-2,3 a io in Figu e S10). This slowe oxida ion a e was
associa ed wi h an inc ease in C alloca ion owa ds anabolism (Djiks a e al., 2001) (Fig-
u e S11).
Fig. S11 Me abolic pa hways o alanine so bed on clay mine als (smec i e and kaolini e),
i on oxides (hama i e and goe hi e) and ac i e coal. De ailed explana ions in
ex . Va ious colo s show he pa hways o C om indi idual posi ions o alanine.
Line wid h ep esen s he quali a i ely es ima ed ela i e shi s in he a e o
alanine C posi ions be ween ce ain pa hways dependen on he so ben class.
Ex ended Summa y
29
Alanine so bed o ac i e coal showed a de ia ing beha io wi h p e e en ial s abili-
za ion o C-3 and oxida ion o C-1 and C-2 (s udy 5, Figu e 3 and 4). This indica es ha in
addi ion o basic mic obial mechanism, u he s abiliza ion and modi ied ans o ma ions
o so bed alanine occu ed, e.g. by exoenzyma ic deg ada ion (Lehmann e al., 2011).
Po en ial deso p ion p ocesses and pa hways a e shown in Figu e S11. In gene al, posi-
ion-speci ic labeling e ealed ha he a e o amino acid C in soil is s ongly a ec ed by
so p ion: The s onge he so p ion, he mo e me abolized LMWOS C is alloca ed in o
mic obial biomass compounds (Figu e S11).
1.3.3.4 Ex a- e sus in acellula ans o ma ion pa hways
Besides in acellula mic obial pa hways, ex acellula ans o ma ions may also
play an impo an ole in LMWOS ans o ma ions in soils, especially in mic ohabi a s
whe e in ac cells ha e no access ( on Lue zow e al., 2006). To dis inguish ex a- and
in acellula ans o ma ion pa hways, selec i e inhibi ion o cellula , ene gy-dependen
pa hways was pe o med in s udy 4 (Figu e S12).
0.02
0.03
0.04
0.05
0 5 10 15 20 25 30 35 40
ime (h)
bio ic Ala-C emo al (µmol)
C-1 Alanine Mic oo ganisms
C-2 Alanine Mic oo ganisms
C-3 Alanine Mic oo ganisms
C-1 Alanine Exoenzymes
C-2 Alanine Exoenzymes
C-3 Alanine Exoenzymes
Fig. S12 Remo al o alanine om soil solu ion by ex a- and in acellula p ocesses wi h-
ou inhibi ion ( illed symbols, dashed line) and by ex acellula ans o ma ion in
espi a ion-inhibi ed ea men s (open symbols, do ed line); Expe imen al poin s
(means ± SEM, N=6) and i ed cu es based on an exponen ial u iliza ion
model a e p esen ed.
This app oach could p o e he exis ence o ex acellula ans o ma ion o LMWOS
in soils: Alanine was decomposed by a s epwise ex acellula oxida ion s a ing om he

Ex ended Summa y
30
ca boxylic g oup, p esumably by a he unspeci ic exoenzymes (Ho ich e e al., 1998).
Howe e , compa ing he kine ics o ex a- e sus in acellula p ocesses (Figu e S12)
e ealed ha cellula up ake o LMWOS always ou -compe ed ex acellula ans o ma-
ions, which a e quan i a i ely ele an only a e y low alanine concen a ions o in spe-
ci ic mic ohabi a s.
1.3.4 Kine ics and ecological ele ance o compe ing sinks
o LMWOS
1.3.4.1 So p ion e sus mic obial u iliza ion
As posi ion-speci ic labeling enables ans o ma ion pa hways in soils o be dis in-
guished, i can be used o assess he ele ance o indi idual pa hways and hei luxes.
So p ion, as an LMWOS sink in soils, can in e ac and compe e wi h mic obial u iliza ion
(Fische e al., 2010b; Kaise and Kalbi z, 2012).
S udy 4 assessed he so p ion o alanine in s e ilized soil and ound ha so p ion
occu ed as in ac molecules and no abio ic clea age o alanine was de ec ed (in con as
o esul s o Wang and Huang (2005)). In s udy 5, he mic obial u iliza ion o so bed
alanine was assessed: Deso p ion, a leas om mine al phases, occu ed mainly as in-
ac molecules, oo. The obse ed posi ion-speci ic ans o ma ions in bo h s udies we e
mainly cha ac e ized by he mic obial me abolism. The e o e, so p ion and deso p ion do
no di ec ly con ibu e o he ans o ma ion o LMWOS, bu only p e en ansloca ion
(Kaise and Kalbi z, 2012) o mic obial ans o ma ion. I kine ics o so p ion and mic obial
up ake we e compa ed (s udy 4), mic obial u iliza ion ou compe ed so p ion in soils.
1.3.4.2 Plan up ake e sus mic obial u iliza ion
Besides mic oo ganisms, plan s a e also known o ha e he abili y o LMWOS up-
ake by hei oo s (Fische e al., 1998). The ele ance o his p ocess is s ill in ques ion
o many ecosys ems due o he e ec i e compe i ion o mic oo ganisms o LMWOS
(Hodge e al., 2000; Jones e al., 2005a). The main p oblem o e alua ing he up ake o
in ac amino acids is me hodological cons ain s: The commonly used dual-iso ope label-
ing app oaches coupled wi h bulk iso ope measu emen s cause an o e es ima ion o he
calcula ed in ac amino acid up ake (Sauhei l e al., 2009). S udy 9 e alua ed he in ac
up ake o he amino acid alanine by plan s using posi ion-speci ic labeling. Consequen ly,
in ac up ake could be dis inguished om he up ake o ans o ma ion agmen s. Posi-
ion-speci ic
14
C-labeling e ealed ha a mino po ion (less han 1.5% o he applied
Ex ended Summa y
31
amino acids) was aken up in ac , whe eas he majo i y o alanine (~98.5%) was used by
soil mic oo ganisms (Figu e S13). Up ake calcula ed om uni o m labeling e lec ed an
o e es ima ion o he ac o 1.2-3 o he quan i ied in ac up ake.
P e e en ial up ake o C-3 by all h ee plan species indica ed ha he up ake o mi-
c obial ans o ma ion agmen s occu ed. P e ious s udies in his hesis e ealed ha
mic obial biomass compounds a e also cha ac e ized by a dominance o alanine C-3
(see s udy 3 and 4). Consequen ly, mic obial up ake and ans o ma ion can p oduce
mine alized N as well as agmen s o he C skele on, which we e pa ially a ailable in he
soil solu ion o oo up ake (Jones e al., 2005). Labeling wi h he N- ee LMWOS ace a e
showed a simila up ake o N-con aining and N- ee LMWOS (< 2% o he applied
14
C).
This indica ed ha plan up ake o LMWOS mainly occu ed ia passi e up ake mecha-
nisms. These passi e mechanisms can also accoun o he unspeci ic up ake o mic o-
bial ans o ma ion p oduc s. In summa y, s udy 9 sugges s ha N up ake om o ganic
sou ces is o mino impo ance o N nu i ion o ag icul u al plan s and e en mo e o he
a e o LMWOS in soils (Jones e al., 2005, Hodge e al., 2000).
Fig. S13 Illus a ion o he a e o alanine (numbe s ep esen % o applied ace : his
can ei he be aken up in ac o deg aded/mine alized o agmen s and subse-
quen ly inco po a ed in o mic oo ganisms o plan biomass).
Ex ended Summa y
32
1.4 Conclusions
The high lux o C h ough he pool o LMWOS clea ly de ines hem as a c ucial C
pools in he SOC cycle. Many p e ious s udies ha e analyzed he a es and u no e o
LMWOS in soil, bu he unde lying mechanisms and pa hways o C ans o ma ion o
LMWOS emains unknown. The e o e, hese s udies we e ocused on biogeochemical
pa hways o h ee main g oups o LMWOS in soil: amino acids, monosaccha ides and
o ganic acids. T acing hei ans o ma ions was achie ed by combining o he i s ime
posi ion-speci ic
13
C-labeling wi h compound-speci ic iso ope analysis (CSIA).
The applica ion o indi idual LMWOS e ealed ha en y s eps in o basic C me-
abolism accoun o speci ics in he C pa i ioning be ween mic obial ca a- and anabo-
lism: subs a es en e ing ci ic acid cycle we e p e e en ially mine alized (>80% in 10
days) whe eas e.g. monosaccha ides en e ing glycolysis we e p e e en ially alloca ed o
anabolic pa hways and inco po a ed in o mic obial biomass (less han 70% mine alized in
10 days).
Posi ion-speci ic
13
C- and
14
C-labeling p o ided a unique submolecula app oach o
econs uc he main pa hways o C ans o ma ion in soil and hei speci ics in indi idual
mic ohabi a s (e.g. a mine al su aces o a he plan - oo in e ace). The di e gence in-
dex (DI) was de eloped and p o en o be a aluable ool o compa e he posi ion-speci ic
a e o indi idual subs ances independen o he used iso opic app oach o expe imen al
design used o he pool in es iga ed.
13
C inco po a ion in a ious mic obial compound
classes was aced by compound-speci ic iso ope analysis: a y acids by GC-C-IRMS
and amino suga s by IC-O-IRMS. The e o e, a new ins umen coupling was applied and
pu i ica ion and measu emen me hods o soil amino suga δ
13
C analysis we e es ab-
lished and e alua ed.
Basic mic obial C me abolism wi h glycolysis, py u a e dehyd ogenase oxida ion
and ci ic acid cycle could be aced in soil unde ield and labo a o y condi ions. Oxidi-
zing, ca abolic pa hways a e ongoing in soils in pa allel o cons uc ing, anabolic pa h-
ways (like gluconeogenesis): o example, up o 55% o he glucose alloca ed o amino
suga syn hesis was no in ac glucose bu de i ed om glucose me aboli es alloca ed by
gluconeogenesis back lux owa ds amino suga o ma ion. Consequen ly, subs a es en-
e ing glycolysis a e in ensi ely ecycled wi hin he cellula C pool, which was shown by a
con inued dec ease o hei di e gence index.
Speci ic ace s o indi idual biosyn he ic pa hways we e iden i ied, which allowed
ans o ma ions o be ollowed wi hin hese side b anches o he basic C me abolism: 1)
he pen ose phospha e pa hway was de ec ed by a combina ion o hexose and pen ose
13
C labeling; and 2) u no e wi hin he cellula lipid pool was p o en by
13
C labeling o
Ex ended Summa y
33
sho - (ace a e) and long-chain (palmi a e) p ecu so s o PLFA. Wi hin 10 days, 65% o
he inco po a ed palmi a e was ans o med (e.g. by desa u a ion, elonga ion o b anch-
ing) o o he a y acids and he inge p in o he palmi a e
13
C-de i ed a y acids ap-
p oached he PLFA pa e n o he p esen mic obial communi y. Knowledge o hese as
a y acid ans o ma ions is c ucial o he applica ion o a y acid inge p in s and hei
iso opic alues o palaeoen i onmen al econs uc ions.
An in ensi e u no e was no only shown o lipids bu also o cell wall polyme s.
Me abolic ecycling ac i i y and u no e was much highe o bac e ia han o ungi,
which was p o en by bo h bioma ke g oups – PLFA and amino suga s. The e o e, his
hesis expe imen ally e ealed one unde lying, mechanis ic eason o he p e iously ob-
se ed speci ics in C u no e o he slow ( ungi-based) and he as (bac e ia-based)
cycling b anch o he soil ood web. Fo he i s ime, posi ion-speci ic labeling was cou-
pled wi h compound-speci ic iso ope analysis o mic obial bioma ke s. This combina ion
p o ides a no el oppo uni y o ace simul aneous, biosyn he ic pa hways o indi idual
mic obial g oups in di e se mic obial communi ies o soils.
Fu he mo e, a ia ions o en i onmen al ac o s, like subs a e concen a ion, we e
iden i ied as he main egula o y ac o s o C alloca ion wi hin mic obial me abolism:
Concen a ion g adien s cha ac e is ic o soils om C-poo bulk soils o ho spo s in-
ol ed a shi o C alloca ion wi hin me abolic pa hways om C s a a ion pa hways ia
main enance pa hways owa ds pa hways ha a e cha ac e is ic o cells unde g ow h
condi ions. So p ion, as a soil-speci ic p ocess educing he bioa ailabili y o a subs a e,
a ec ed mic obial me abolism: he s onge a subs a e is so bed, he mo e o i s C is
alloca ed owa ds anabolism, e.g. is ound in he mic obial p oduc s. Unde s anding hese
shi s in me abolic pa hways is c ucial o he SOC cycle, as C alloca ion owa ds anabo-
lism is he p e equisi e o he o ma ion and s abiliza ion o mic obially-de i ed SOM.
Th ee soil-speci ic p ocesses we e aced in pa allel wi h mic obial u iliza ion: so p-
ion, exoenzyma ic LMWOS u iliza ion and plan up ake. So p ion, as well as deso p ion,
occu ed as in ac molecules and did no accoun o LMWOS ans o ma ions. Exoen-
zymes caused a s epwise oxida ion o he LMWOS C backbone. Howe e , hei kine ics
could no compe e wi h mic obial up ake sys ems. Consequen ly, ex acellula ans o -
ma ions can only be ele an in speci ic soil mic ohabi a s, which a e inaccessible o
mic oo ganisms. In ac up ake o amino acids by plan s was assessed by dual-iso ope
posi ion-speci ic
13
C- and
15
N-labeling. This new app oach e ealed he o e es ima ion o
in ac amino acid up ake due o me hodological cons ain s o p e ious s udies and
showed ha less han 1.5% o he applied amino acids we e aken up in ac by plan s.
Consequen ly, none o he in es iga ed abio ic o bio ic p ocesses could compe e wi h
Ex ended Summa y
40
1.6 Con ibu ion o he included manusc ip s and
publica ions
The publica ions and manusc ip s included in his PhD hesis we e p epa ed in coope a-
ion wi h a ious coau ho s. The coau ho s lis ed in hese publica ions and manusc ip s
con ibu ed as ollows:
S udy 1:
Fa e o low molecula weigh o ganic subs ances in an a able soil: om mic obial up ake
o u ilisa ion and s abilisa ion
S a us a da e o hesis submission: Submi ed o Soil Biology and Biochemis y;
S a us a da e o hesis p in ing: Submi ed o Soil Biology and Biochemis y;
Con ibu o s:
Anna Gunina 45% Accomplishmen o expe imen s, labo a o y analysis,
da a p epa a ion, p epa a ion o he manusc ip
Michaela Dippold 40% Expe imen al design, accomplishmen o he expe i-
men s, da a p epa a ion, p epa a ion o he manusc ip
B uno Glase 3% Discussions on expe imen al design, sugges ions o
imp o e manusc ip
Yako Kuzyako 12% Expe imen al design, discussions on he esul s, sug-
ges ions o imp o e manusc ip
S udy 2:
Imp o ed δ
13
C analysis o amino suga s in soil by Ion Ch oma og aphy - Oxida ion - Iso-
ope Ra io Mass Spec ome y
S a us a da e o hesis submission: In e ision a Rapid Communica ions in Mass
Spec ome y;
S a us a da e o hesis p in ing: Published in Rapid Communica ions in Mass Spec o-
me y;
Con ibu o s:
Michaela Dippold 55% Es ablishmen o pu i ica ion me hod
, labo a o y analysis,
da a p epa a ion, p epa a ion o he manusc ip
S e anie Boesel 20% Es ablishmen o measu emen me hod, sugges ions o
imp o e manusc ip
Anna Gunina 12% Labo a o y analysis, commen s o imp o e manusc ip

Ex ended Summa y
41
Yako Kuzyako 3% Sugges ions o imp o e manusc ip
B uno Glase 10% Discussions on me hod de elopmen , sugges ions o
imp o e manusc ip
S udy 3:
Biochemical pa hways o amino acids in soil: Assessmen by posi ion-speci ic labeling
and
13
C-PLFA analysis
S a us a da e o hesis submission: Accep ed in Soil Biology and Biochemis y
S a us a da e o hesis p in ing: Published in Soil Biology and Biochemis y
Con ibu o s:
Ca olin Apos el 42% Accomplishmen o expe imen s, labo a o y analysis,
da a p epa a ion, p epa a ion o he manusc ip
Michaela Dippold 40% Expe imen al design, accomplishmen o he expe i-
men s, da a p epa a ion, sugges ions o imp o e manu-
sc ip
B uno Glase 3% Labo a o y analysis, sugges ions o imp o e manusc ip
Yako Kuzyako 15% Expe imen al design, discussions on he esul s, sug-
ges ions o imp o e manusc ip
S udy 4:
Biogeochemical ans o ma ions o amino acids in soil assessed by posi ion-speci ic
labelling
S a us a da e o hesis submission: Accep ed in Plan and Soil
S a us a da e o hesis p in ing: Published in Plan and Soil
Con ibu o s:
Michaela Dippold 85% Expe imen al design, accomplishmen o expe imen ,
da a p epa a ion, p epa a ion o he manusc ip
Yako Kuzyako 15% Expe imen al design, discussions on he esul s, sug-
ges ions o imp o e manusc ip
S udy 5:
So p ion a ec s amino acid pa hways in soil: Implica ions om posi ion-speci ic labeling
o alanine
S a us a da e o hesis submission: Submi ed o Soil Biology and Biochemis y
S a us a da e o hesis p in ing: Accep ed in Soil Biology and Biochemis y
Ex ended Summa y
42
Con ibu o s:
Michaela Dippold 60% Expe imen al design, da a p epa a ion, p epa a ion o
he manusc ip
Mikhail Bi yuko 25% Accomplishmen o expe imen , da a p epa a ion,
Yako Kuzyako 15% Expe imen al design, discussions on he esul s, sug-
ges ions o imp o e manusc ip
S udy 6:
Biochemis y o hexose and pen ose ans o ma ions in soil analyzed by posi ion-speci ic
labeling and
13
C-PLFA
S a us a da e o hesis submission: To be submi ed o Soil Biology and Biochemis y
S a us a da e o hesis p in ing: Resubmi ed o Soil Biology and Biochemis y
Con ibu o s:
Michaela Dippold 45% Expe imen al design, accomplishmen o expe imen s,
da a p epa a ion, p epa a ion o he manusc ip
Ca olin Apos el 45% Accomplishmen o expe imen , da a p epa a ion,
p epa a ion o he manusc ip
Yako Kuzyako 10% Discussions on he esul s, sugges ions o imp o e
manusc ip
S udy 7:
Me abolic pa hways o ungal and bac e ial amino suga o ma ion in soil assessed by
posi ion-speci ic
13
C-labeling
S a us a da e o hesis submission: To be submi ed o Biogeochemis y
S a us a da e o hesis p in ing: Submi ed o Soil Biology and Biochemis y
Con ibu o s:
Michaela Dippold 60% Expe imen al design, accomplishmen o expe imen s,
pa a ion, p epa
da a p epa a ion, p epa a ion o he manusc ip
Anna Gunina 20% Labo a o y analysis, sugges ions o imp o e manusc ip
S e anie Boesel 5% Labo a o y analysis, da a p epa a ion
B uno Glase 3% Sugges ions o imp o e manusc ip
Yako Kuzyako 12% Discussions on expe imen al design, sugges ions o
imp o e manusc ip
Ex ended Summa y
43
S udy 8:
Fo ma ion and ans o ma ion o a y acids in soils assessed by posi ion-speci ic labeling
o p ecu so s
S a us a da e o hesis submission: To be submi ed o O ganic Geochemis y
S a us a da e o hesis p in ing: Submi ed o Geochimica e Cosmochimica Ac a
Con ibu o s:
Michaela Dippold 85% Expe imen al design, accomplishmen o expe imen ,
da a p epa a ion, p epa a ion o he manusc ip
Yako Kuzyako 15% Expe imen al design, discussions on he esul s, sug-
ges ions o imp o e he manusc ip
S udy 9:
O ganic N up ake by plan s - Ree alua ion by posi ion-speci ic labeling o amino acids
S a us a da e o hesis submission: Submi ed o Soil Biology and Biochemis y
S a us a da e o hesis p in ing: Submi ed o Jou nal o Expe imen al Bo any
Con ibu o s:
Daniel Mo an 42% Accomplishmen o expe imen , da a p epa a ion,
p epa a ion o he manusc ip
Michaela Dippold 42% Expe imen al design, accomplishmen o expe imen ,
da a p epa a ion, p epa a ion o he manusc ip
B uno Glase 3% Labo a o y analysis, sugges ions o imp o e he manu-
sc ip
Yako Kuzyako 13% Expe imen al Design, Discussions on he esul s, sug-
ges ions o imp o e he manusc ip
Publica ions and Manusc ip s
44
2 Publica ions and Manusc ip s
2.1 S udy 1: Fa e o low molecula weigh o ganic
subs ances in an a able soil: om mic obial up-
ake o u ilisa ion and s abilisa ion
Anna Gunina
1,2,3
, Michaela A. Dippold
1,2
, B uno
Glase
4
, Yako Kuzyako
1,5
1
Depa men o Ag icul u al Soil Science, Geo g-Augus -Uni e si y o Gö ingen
2
Depa men o Ag oecosys em Resea ch, Uni e si y o Bay eu h
3
Facul y o soil science, Moscow Lomonoso S a e Uni e si y
4
Depa men o Soil Biogeochemis y, Ins i u e o Ag icul u al and Nu i ional Science,
Ma in-Lu he Uni e si y Halle-Wi enbe g
5
Depa men o Soil Science o Tempe a e Ecosys ems, Geo g-Augus -Uni e si y o
Gö ingen
Co esponding Au ho :
Anna Gunina
Max Planck Ins i u e o Biogeochemis y
Am He enbe ge 11
07745 Jena
email: guninaa[email p o ec ed]m
Tel.: 0157/85566093
Publica ions and Manusc ip s
45
Abs ac
Mic obial up ake and u iliza ion a e he main ans o ma ion pa hways o low mo-
lecula weigh o ganic subs ances (LMWOS) in soil, bu de ailed o ans o ma ions is
s ongly limi ed. As a ious LMWOS classes en e biochemical cycles a di e en s eps,
we hypo hesize ha he pe cen age o hei ca bon (C) used by mic obial biomass and
consequen ly s abiliza ion in soil is di e en .
Rep esen a i es o he h ee main g oups o LMWOS: amino acids (alanine, glu a-
ma e), suga s (glucose, ibose) and ca boxylic acids (ace a e, palmi a e) – we e applied
a na u ally-occu ing concen a ions in o a loamy a able Lu isol in a ield expe imen .
Inco po a ion o
13
C om hese LMWOS in o mic obial biomass (MB) and in o phosphol-
ipid a y acids (PLFAs) was in es iga ed 3 d and 10 d a e applica ion. The mic obial
u iliza ion o LMWOS o cell memb ane cons uc ion was es ima ed by eplacemen o
PLFA-C wi h
13
C.
Mine aliza ion o LMWOS o CO
2
comp ised 20–65% o he ini ially applied
13
C,
whe eas
13
C inco po a ion in o MB amoun ed o 10–24% a day 3 and was educed o 1–
15% on day 10. Maximal inco po a ion o
13
C in o MB was obse ed om suga s and
minimal om amino acids. S ong di e ences in mic obial u iliza ion be ween LMWOS
we e obse ed mainly a day 10. Thus, despi e simila ini ial apid up ake by mic oo gan-
isms, u he me abolism wi hin mic obial cells accoun s o he speci ic a e o C om
a ious LMWOS in soils.
13
C om each LMWOS was inco po a ed in o each PLFA. This e lec s he ubiqui-
ous u iliza ion o all LMWOS by all unc ional mic obial g oups. The p e e en ial inco po-
a ion o palmi a e in o PLFAs e lec s i s ole as a di ec p ecu so o a y acids. Highe
13
C inco po a ion om alanine and glucose in o PLFAs compa ed o glu ama e, ibose
and ace a e e lec s he p e e en ial use o glycolysis-de i ed subs ances in he a y ac-
ids syn hesis.
G am-nega i e bac e ia (16:1ω7c and 18:1ω7c) we e he mos abundan and ac i e
in LMWOS u iliza ion. Thei high ac i i y co esponds o a high demand o anabolic
p oduc s, e.g. o dominance o pen ose-phospha e pa hway, i.e. inco po a ion o ibose-C
in o PLFAs. The
13
C inco po a ion om suga s and amino acids in ilamen ous mic oo -
ganisms was lowe han in all p oca yo ic g oups. Howe e , o ca boxylic acids, he in-
co po a ion was in he same ange (0.1 – 0.2% o he applied ca boxylic acid C) as ha o
g am-posi i e bac e ia. This may e lec he dominance o ungi and o he ilamen ous
mic oo ganisms o u iliza ion o acidic and complex o ganics.
Thus, we showed he di e gence o C pa hways om LMWOS o e he 10 days,
despi e hei simila ini ial up ake by mic oo ganisms. Consequen ly, s abiliza ion o C in

Publica ions and Manusc ip s
46
soil is mainly connec ed no wi h i s ini ial mic obial up ake, bu wi h i s inco po a ion in o
mic obial compounds o a ious s abili y.
Keywo ds: low molecula weigh o ganic subs ances,
13
C-labelling, monosaccha ides,
amino acids, ca boxylic acids, compound-speci ic iso ope analysis, PLFAs u no e , soil
mic oo ganisms
Publica ions and Manusc ip s
47
2.1.1 In oduc ion
Low molecula weigh o ganic subs ances (LMWOS) comp ise 5–10% o dissol ed
o ganic ca bon (DOC) in soils (Ryan e al., 2001) and a e p oduc s o hizodeposi ion,
abo e and belowg ound li e and mic obial esidue deg ada ion.
Mic obial emo al o LMWOS om solu ion in he uppe soil ho izons appea s
wi hin minu es (Jones e al., 2004), whe eas he hal -li e o C om LMWOS is much
longe , om se e al hou s o mon hs o e en decades ( an Hees e al., 2005). This oc-
cu s due o apid mic obial up ake und u he u ilisa ion o LMWOS wi hin he mic obial
biomass, which ou -compe e p ocesses o physicochemical so p ion o LMWOS a min-
e al su aces and hei leaching om he soil p o ile, p obably by o de s o magni ude
(Fische e al., 2010). Due o he s ong link LMWOS dynamics wi h mic obial u iliza ion,
he a e o LMWOS should be in es iga ed a na u al applied amoun s, o a oid any
changes in mic obial esponse s a egy.
The main compound classes wi hin he LMWOS a e amino acids, suga s (mainly
monosaccha ides) and ca boxylic acids (Fische e al., 2010). Amino acids ep esen he
la ges pool o N in soils, mainly bound in p o eins. Abou 30% o N ob ained a e acid
hyd olysis om he p o ein pool (S e enson, 1982) and a la ge po ion o N eleased om
soil o ganic ma e (SOM) by enzymes a e amino acids (Ba aclough, 1997). Amino acid-
C hal -li es a e be ween 3–45 days and do no s ongly di e be ween ield and labo a-
o y condi ions (Glan ille e al., 2012). The g ea a iabili y in amino acid u ilisa ion e-
po ed in he li e a u e is a consequence o he di e si y o me abolic pa hways wi hin
mic obial cells (Apos el e al., 2013) and also can be depend on ac i i y o mic oo gan-
isms (Jones e al., 2005).
Nume ous s udies ha e epo ed ha ca bohyd a es a e he mos abundan sub-
s ance class, amoun ing o 5–25% o soil o ganic ma e (SOM) (Benzingpu die, 1980;
Cheshi e, 1979). Glucose is he mos abundan ca bohyd a e de i ed ei he om he de-
composi ion o plan esidues (De ien e al., 2006) o om oo exuda es (De ien e al.,
2004; Fische and Kuzyako , 2010). Hal -li e o glucose-de i ed C comp ises a ound 15
days in ield condi ions (Glan ille e al., 2012). On a e age, 60% o he added glucose is
inco po a ed in o cellula compounds (Fische e al., 2010) Despi e glucose supposed o
be a ubiqui ous subs a e, which can be used by nea ly all mic oo ganisms (Macu a and
Kuba o a, 1973), speci ics o i s u ilisa ion in soil is s ill a opic o discussion (Reischke e
al., 2014).
The hi d mos abundan class o LMWOS in soils is ca boxylic acids. 80–90% o C
om he applied ca boxylic acids we e decomposed du ing he i s 7 days and only 10–
20% o C we e inco po a ed in o mic obial biomass (MB) (Un e egelsbache e al., 2012;
Publica ions and Manusc ip s
48
an Hees e al., 2002). The u ilisa ion o ca boxylic acids is subs a e-dependen : ace a e
has a lowe mine aliza ion capaci y han ci a e and oxala e ( an Hees e al., 2002) and
ci a e can be deg aded as e han mala e and oxala e (S om e al., 2001). A he in-
amolecula le el, –COOH g oups can be oxidized o CO
2
e y apidly, whe eas CH
3
-
g oups a e p e e en ially used o biosyn hesis (Dippold and Kuzyako , 2013; Fische and
Kuzyako , 2010). Thus, di e ences in ca boxylic acid u ilisa ion can be a ibu ed o hei
a ious oles in cell me abolism as well as o hei di e ences in chemical s uc u e.
S udies ha simul aneously compa e he a e o amino acids, suga s and ca boxylic
acids a e no nume ously epo ed in li e a u e. In mos cases s udies ei he conside he
mic obial u ilisa ion o LMWOS by he en i e MB (Glan ille e al., 2012; Rousk and Baa h,
2011) o ocus on con ibu ion o a ious mic obial g oups o LMWOS u iliza ion (Apos el
e al., 2013; Rinnan and Båå h, 2009; Rinnan e al., 2013).
In o ma ion conce ning he e alua ion o he con ibu ion o unc ional mic obial
g oups o LMWOS u iliza ion can be ob ained by coupling o
13
C o
14
C labeling wi h
analysis o mic obial bioma ke s such as amino suga s (Amelung e al., 2001; Engelking
e al., 2007; Glase e al., 2004), phospholipids-de i ed a y acids (F os ega d e al.,
2011; Zelles, 1997) o DNA-based me hods (Ibekwe e al., 2002, Radejewski e al. 2003).
Coupling PLFA analysis wi h
13
C-labelling has shown ha g am-nega i e (G-) bac e ia
a e mo e ac i e in he u ilisa ion o plan C (low o high molecula weigh ) han g am-
posi i e (G+) bac e ia, e en i he la e g oup has a highe PLFA con en in soil (Ga cia-
Pausas and Pa e son, 2011; Wald op and Fi es one, 2004). Fungi con ibu e less o he
u ilisa ion o plan -de i ed C han bac e ia (Wald op and Fi es one, 2004). In con as , he
use o
13
C pulse-labelling o plan s o ace
13
C in PLFAs has shown ha ei he ungi
(Bu le e al., 2003) o G- bac e ia (Tian e al., 2013) a e he mos ac i e in hizodeposi s
consume . Inco po a ion o
13
C om labelled s aw in o PLFAs has shown ha a y acids
such as 16:0; 18:1w9, 18:2w6,9 we e mo e
13
C-en iched, whe eas o he 16:1w5 o
10Me17:0 a y acids con ained negligible amoun s o
13
C (Williams e al., 2006). Conse-
quen ly, membe s o he mic obial communi y a e di e en ially in ol ed in he assimila-
ion o li e - o oo -de i ed C (Williams e al., 2006) and he ac i i y o indi idual mic o-
bial g oups appea s o depend on he quali y o subs a e and on en i onmen al condi-
ions such as soil ype, season and clima ic condi ions (B ay e al., 2012). Thus, gene al
p inciples o LMWOS u ilisa ion by indi idual g oups o bac e ia and ungi s ill emain
open.
The second ac o con olling LMWOS a e is mic obial me abolism: a ious classes
o LMWOS en e di e en pa hways and consequen ly a e u ilised di e en ly. Suga s a e
mainly used di ec ly by he basic glycolysis pa hway (Caspi e al., 2008; Kesele e al.,
2009), ca boxylic acids en e om side b anches o he ci ic acid cycle (Caspi e al.,
Publica ions and Manusc ip s
49
2008; Kesele e al., 2009), and amino acids en e glycolysis o he ci ic acid cycle om
indi idual side b anches a di e en s eps (Apos el e al., 2013; Knowles e al., 2010).
Thus, we assume ha uni e sal subs ances such as suga s, en e ing glycolysis di ec ly,
will be me abolised e y apidly in compa ison o ca boxylic acids and amino acids en e -
ing he ci ic acid cycle. Howe e , glycolysis also enables en y in o many anabolic pa h-
ways, i.e., we hypo hesise ha suga s a e used mo e o anabolism han ca boxylic ac-
ids, which en e he oxidising ci ic acid cycle and can be di ec ly me abolised o ene gy
p oduc ion. The highes di e si y in pa hways can be expec ed o amino acids, because
hey en e basic me abolism a a ious s eps (Apos el e al., 2013). Since ca boxylic acid
u ilisa ion is subs a e-con olled, we expec di e gence in he u ilisa ion o sho - and
long-chain acids. Because h ee classes o LMWOS en e me abolic cycles a a ious
poin s, we hypo hesise ha hei ole in he syn hesis o cell componen s such as PLFAs
should be di e en .
Thus, he o e all aim o his s udy was o es ima e he sho - e m ans o ma ion o
ep esen a i es o h ee main classes o LMWOS: monosaccha ides (glucose and i-
bose), ca boxylic acids (ace a e and palmi a e) and amino acids (alanine and glu ama e)
unde ield condi ions, coupling
13
C subs a e labelling wi h he analysis o speci ic cell
PLFA bioma ke s.
2.1.2 Ma e ial and Me hods
2.1.2.1 Expe imen al design
The ield expe imen was ca ied ou a an ag icul u al ield ial in Hohenpölz
(49°54' N, 11°08'E, a 500 m a.s.l.). The mean annual empe a u e was +7
o
C and mean
annual p ecipi a ion was 870 mm. The si e is cul i a ed by a o a ion o i icale, whea
and ba ley. The soil is an a able loamy haplic Lu isol (IUSS Wo king g oup WRB, 2007)
and had he ollowing cha ac e is ics: pH 6.6, o al C con en 1.5%, C/N 10.7, CEC 13
cmol
C
kg
-1
, clay con en 22%.
In Augus 2010, ollowing ha es o he i icale and spudding o he soil, columns
we e inse ed o a dep h o 10 cm and six,
13
C uni o mly-labelled subs ances: alanine,
glu ama e, glucose, ibose, sodium ace a e and palmi a e we e injec ed in o sepa a e
columns. The amoun s o applied ace we e: alanine 96.3, glu ama e 91.6, glucose
93.4, ibose 91.8, ace a e 95.8 and palmi a e 49.5 µmol
13
C column
-1
. The amoun o
added C was kep as low as possible and cons an o all columns, including he con ols,
whe e simila amoun s o non-labelled C was applied (0.40–0.77 µg C g soil
-1
). Each col-
umn con ained 1.5 kg soil. The ield expe imen had a andomised block design wi h ou
Publica ions and Manusc ip s
56
Fig. 2
13
C inco po a ion om bo h amino acids (in % o applied
13
C) in o PLFAs ( op)
and pe cen o
13
C eplacemen (in % o PLFA-C) (bo om) o mic obial g oups 3
and 10 days a e alanine and glu ama e applica ion. Le e s e lec signi ican
di e ences be ween alanine and glu ama e up ake in o mic obial g oups.
U ilisa ion o
13
C om suga s o PLFAs o ma ion showed di e en ends in bac e-
ial and ungal g oups and much highe absolu e
13
C inco po a ion compa ed o amino
acids (Fig. 3, op). Be ween 0.01 and 0.70% o ini ially applied suga s
13
C was ound in
a ious axonomic g oups a e h ee days, whe eas only 0.001–0.25% was eco e ed
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Alanine
Glu ama e
Inco po a ion (% o applied
13C)
G-1 G-2 G+1 G+2 G+3 Ac 16:1w5 F P
a
b
a
a
a
b
b
b
Inco po a ion
0
0.02
0.04
0.06
a
b
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
3 10 3 10 3 10 3 10 3 10 3 10 3 10 3 10 3 10
Days a e LMWOS applica ion
Replacemen by
13
C (% o PLFA-C)
a
b
a
b
a
a
a
b
b
b
b
Replacemen
a

Publica ions and Manusc ip s
57
om amino acids. The inco po a ion o
13
C om suga s in o all mic obial g oups in-
c eased o emained cons an be ween days h ee and 10. All bac e ial species used
glucose-C mo e e icien ly han ibose-C excep G- g oup 1, which p e e ed ibose.
Among he ilamen ous mic oo ganisms, ungi did no di e om 16:1w5 in glucose-
13
C
inco po a ion in o PLFAs, bu ungi used mo e
13
C om ibose han 16:1w5. In gene al,
he mic obial specialisa ion o indi idual monosaccha ides as building blocks o PLFAs
was isible wi hin bac e ial bu no wi hin euka yo ic g oups.
Fig. 3
13
C inco po a ion om bo h monosaccha ides (in % o applied
13
C) in o PLFAs
( op) and pe cen o
13
C eplacemen (in % o PLFA-C) (bo om) o mic obial
g oups 3 and 10 days a e glucose and ibose applica ion. Le e s e lec sig-
ni ican di e ences be ween glucose and ibose up ake in o mic obial g oups
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Glucose
Ribose
Inco po a ion (% o applied
13
C)
G-1 G-2 G+1 G+2 G+3 Ac 16:1w5 F P
Inco po a ion
a
b
a
b
a
b
0
0.02
0.04
0.06
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0.22
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
3
10
Replacemen by
13
C (% o PLFA-C)
Days a e LMWOS applica ion
Replacemen
a
b
a
a
b
b
a
b
Publica ions and Manusc ip s
58
Inco po a ion o
13
C om bo h ca boxylic acids in o PLFAs o G- bac e ia g oup 1
was highe han om o he LMWOS (Fig. 4, op). O he bac e ial g oups used
13
C om
ca boxylic acids less e icien ly han
13
C om suga s and amino acids o PLFAs syn he-
sis. Filamen ous mic oo ganisms (ac inomyce es and ungi, bu also 16:1w5) exceeded
he p oka yo ic g oups o G+ bac e ia in inco po a ion o
13
C om he mos complex sub-
s a e, palmi a e, in o PLFAs.
Fig. 4
13
C inco po a ion om bo h ca boxylic acids (in % o applied
13
C) in o PLFAs
( op) and pe cen o
13
C eplacemen (in % o PLFA-C) (bo om) o mic obial
g oups 3 and 10 days a e ace a e and palmi a e applica ion. Le e s e lec
signi ican di e ences be ween ace a e and palmi a e up ake in o mic obial
g oups.
0
0.05
0.1
0.15
0.2
Ace a e
Palmi a e
Inco po a ion (% o applied
13
C)
G-1 G-2 G+1 G+2 G+3 Ac 16:1w5 F P
Inco po a ion
a
a
bb
b
a
a
b
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
3 10 3 10 3 10 3 10 3 10 3 10 3 10 3 10 3 10
Days a e LMWOS applica ion
Replacemen by
13
C (% o PLFA-C)
Replacemen
a
b
a
a
a
b
b
b
a
a
b b
0
0.5
1
a
b
Publica ions and Manusc ip s
59
In gene al, inco po a ion o LMWOS-C in bac e ial species was highe han ha in
euka yo es.
2.1.4 Discussion
2.1.4.1
Inco po a ion o LMWOS in o SOM and mic obial biomass
Amino acids
The mine aliza ion o alanine and glu ama e in ou expe imen was simila o li e a-
u e da a (Jones e al., 2005) and less han 50% o applied
13
С
emained in he soil a e
10 days. The hal -li e o alanine and glu ama e-de i ed C epo ed o ield condi ions was
18 and 3 days, espec i ely (Glan ille e al., 2012). This is much longe han in ou ex-
pe imen and we obse ed a simila mine aliza ion o glu ama e and alanine C wi hin 10
days. These con as ing esul s migh be a ibu able o di e ences in o al mic obial ac-
i i y o communi y s uc u e (Jones e al., 2005) in he s udied soils as well as due o
me hodological di e ences. Simila
13
C amoun s om glu ama e and alanine emaining in
he soil on day 10 e lec ed a simila mine aliza ion o hese amino acids and co esponds
o simila mic obial decomposi ion o di e en ly cha ged amino acids (Jones and Hodge,
1999).
The high amoun o
13
C inco po a ed in o he EMB pool a day h ee (Fig. 1) co e-
sponded wi h he apid and e icien up ake o ee amino acids as in ac molecules
(Dippold and Kuzyako , 2013; Geissele e al., 2010; Jones and Hodge, 1999). A e up-
ake, amino acids can ei he be oxidised o ene gy p oduc ion, di ec ly inco po a ed in o
p o eins (Geissele e al., 2010) o used in o he me abolic pa hways (Fig. 5) (Dippold
and Kuzyako , 2013; Knowles e al., 2010). The inco po a ion o alanine in o EMB was
highe han o glu ama e on day 10, showing he mo e apid mine alisa ion o glu ama e
C. Simila ly, glu ama e was u ilised mo e apidly han glycine and lysine o e a b oad
concen a ion ange (Jones and Hodge, 1999). This co esponds o he di e en en y
poin o hese amino acids in o me abolism (Knowles e al., 2010). Alanine en e s he
basic cellula me abolism a he connec ing s ep be ween glycolysis and he ci ic acid
cycle (Apos el e al., 2013; Caspi e al., 2008). Thus, i is easily dis ibu ed h oughou all
anabolic pa hways o he syn hesis o cell componen s, e.g., glyconeogenesis, p o ein
syn hesis, a y acid syn hesis and ibonucleo ide syn hesis (Fig. 5). In con as , glu a-
ma e di ec ly en e s he ci ic acid cycle as oxoglu a a e (Knowles e al., 2010). This de-
mands ene gy o glyconeogenesis and he e o e, a y acid syn hesis pa hways will no
be used i o he mo e app op ia e subs a es a e a ailable. Thus, alanine C is p e e en-
ially inco po a ed in o he mo e s able componen s o mic obial cells – he cell walls and
Publica ions and Manusc ip s
60
he memb anes – compa ed o glu ama e. In con as , glu ama e plays a cen al ole in
he amino acid cycle, and oxoglu a a e p oduced om glu ama e by ansamina ion will
be apidly decompose o CO
2
(Vinolas e al., 2001).
Fig. 5 P ima y me abolic pa hways o he six ep esen a i es o h ee LMWOS classes
(amino acids (blue), suga s (g een) and ca boxylic acids ( ed)). Thick a ows e-
lec he en e ing poin s o LMWOS in he me abolic pa hways; black ine a ows
show he basic C me abolism and shaded a ows e lec anabolic pa hways o
o ma ion o cellula compounds.
Suga s
The hal -li e o glucose-C in ou expe imen (25% mine alized wi hin 10 days) is
wi hin he ange o p e ious s udies: Glan ille e al. (2012) epo ed a decomposi ion o
50% o glucose-C a e 20 days, Sagga e al. (1999) measu ed a glucose-C decomposi-
ion o 51–66% wi hin 35 days and Schneckenbe ge e al. (2008) obse ed a mine alisa-
ion o 26–44% o
14
C om glucose wi hin 22 days. The inco po a ion o a signi ican p o-
po ion o applied 13C om suga s in o EMB in ou expe imen is in ag eemen wi h he
model o sho - e m glucose u ilisa ion (Nguyen and Gucke , 2001). In his model, glu-
cose aken up om solu ion is ini ially alloca ed o an in e media e pool and he ea e
can be espi ed o used as a s uc u al componen . Thus, due o he demand o cellula
p oduc s, glucose C will p e e en ially be ans e ed o anabolic pa hways a he han be
oxidised o ene gy p oduc ion.
Publica ions and Manusc ip s
61
The lowe mine alisa ion and inco po a ion o pen ose compa ed o hexose on day
h ee co esponds wi h i s slowe up ake a e, as i is a less common soil monosaccha-
ide han glucose. The me abolisa ion o pen oses occu s mainly ia he pen ose-
phospha e-pa hway, leading o inco po a ion in o a ious cell componen s such as DNA
o o he ibonucleo ides (Fig. 5). Bo h phenomena explain he lowe u ilisa ion o ibose
compa ed o glucose. P e e en ial inco po a ion o ibose in o biosyn he ic ibonucleo ide
p oduc s has ye o be p o en by subs ance-speci ic analysis, e.g., by s able iso ope
p obing o DNA (Radajewski e al., 2000). Indeed, ibose mine alisa ion and inco po a ion
in o he EMB on day 10 we e nea ly he same as o glucose. This con i ms ha he hex-
ose and pen ose pa hways a e closely linked and ha C om bo h monosacca ides is
ans e ed wi hin hese pa hways owa ds biosyn he ic pa hways acco ding o he C de-
mand o cells.
Ca boxylic acids
Simila o amino acids and monosaccha ides, he apid up ake o ace a e ou com-
pe es i s physicochemical so p ion in soils (Fische e al., 2010). Wi hin he mic obial
biomass, ace a e C can be subjec ed o “a es me abolism” and s o ed in cells be o e
use (Fische and Kuzyako , 2010). Ace a e can also be ans o med in o ca bohyd a es,
amino acids (So ensen and Paul, 1971) and o he cell componen s and hus is ixed in
di e se mic obial p oduc s. Inco po a ion o ace a e in o EMB was less han ha o sug-
a s, which con i ms ha ace a e was used o espi a ion (ca. 80–90%) a he han o
new cell biomass p oduc ion (Jones and Edwa ds, 1998; an Hees e al., 2002). This
occu s due o he oxida ion o a high p opo ion o ace a e C in o he ci ic acid cycle. Fu -
he mo e, ace a e mus be ac i a ed p io o inco po a ion in o he key me abolic pa h-
ways ( an Hees e al., 2002). The ans o ma ion o ace a e o anabolic p oduc s is hus
un a o able, as long as mic oo ganisms ha e access o mo e eely a ailable subs a es.
An excep ion o his pa hway, howe e , is a y acid syn hesis, whe e ace a e is a di ec
p ecu so .
Palmi a e is an anion o a sho -chain a y acid, he mos dominan a y acid in
bac e ia and ungi (Lawlo e al., 2000) and is a p ecu so o he syn hesis o mo e com-
plex a y acids. Due o i s high molecula weigh and long alipha ic chain, we hypo he-
sised ha i s decomposi ion is much slowe han he decomposi ion o sho chain ca -
boxylic acids such as ace a e. Howe e , his hypo hesis was no con i med in ou ex-
pe imen s. P e ious s udies showed ha he deg ada ion o palmi a e was mo e apid
han ha o simila o longe a y acids (Moucawi e al., 1981). I was es ima ed ha 41%
o oleic and 31% o s ea ic acids we e decomposed wi hin ou weeks (Moucawi e al.,
1981), whe eas 50% o palmi a e decomposed wi hin 10 days (Fig. 1).

Publica ions and Manusc ip s
62
In con as o ne mine aliza ion, he inco po a ion o
13
C om palmi a e in o EMB
was he lowes (Fig. 1). This migh be due o hyd ophobic in e ac ions o palmi a e wi h
SOM ha led o he lowe up ake in o mic obial biomass compa ed o o he LMWOS.
Howe e , i aken up, i was p e e en ially inco po a ed in PLFAs, bu no used o he
syn hesis o o he mic obial compounds. Inco po a ion in o PLFAs was highe han o
any o he LMWOS, which is in acco dance wi h hei di ec p ecu so ole o PLFA o -
ma ion.
In gene al, ou esul s e lec ha he up ake and u ilisa ion o six LMWOS wi hin 3
days was qui e simila and compa able wi h he li e a u e (Glan ille e al., 2012). As a
as subs ance-speci ic di e ences in inco po a ion in o EMB we e he mos isible only a
day 10 (Supplemen a y Table 3.), when he o al amoun o inco po a ed C dec eased,
he long- e m a e o LMWOS-C in soils a e caused by he me abolic pa hways o
LMWOS classes wi hin mic obial cells and no by apid LMWOS up ake du ing he i s
ew days. The e o e, we can conside he medium- e m di e gence o C depending on i s
ini ial o m ha en e ed he soil.
2.1.4.2
Mic obial communi y composi ion
The cons an composi ion o PLFAs (Table 1) a e he addi ion o e y low amoun
o LMWOS-C shows ha mic obial communi y s uc u e was unde s eady-s a e condi-
ions (Blagoda skaya e al., 2007, , 2009). This co esponds o o he s udies wi h simila
amoun s o applied C (B an e al., 2006) and leads o
13
C inco po a ion in o mic obial
biomass and indi idual mic obial g oups e lec ed he ypical u iliza ion o hese sub-
s ances unde na u al soil condi ions – i.e. a mic oo ganisms unde main enance me abo-
lism.
The main classes o decompose s o he six subs ances we e G- and G+ bac e ia.
G- bac e ia a e e y common in he hizosphe e, which e lec s hei p e e ence o
LMWOS common in hizosphe e ho spo s. In con as , G+ bac e ia a e abundan in bulk
soil (Sode be g e al., 2004).
The soil en i onmen o his s udy, wi h ae obic condi ions, a neu al soil pH as well
as he abo e- and belowg ound li e emaining a e he ha es , p o ide op imum condi-
ions o he de elopmen o ac inomyce es, which is assumed o be impo an o he
p ima y deg ada ion o ecalci an SOM (McCa hy and Williams, 1992). In con as , p e-
sen en i onmen al condi ions and loadings o small amoun s o complex subs a es did
no suppo ed ungal g ow h (Reischke e al., 2014), which explains hei low abundance
as well as hei low ac i i y in LMWOS u iliza ion in ou expe imen .
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63
We de ec ed ele an amoun s o 16:1w5 a y acid, which can be used o cha ac-
e ized he VAM ungi o g am-nega i e bac e ia (Olsson, 1999; Zelles, 1997). Resul s o
he ac o analysis do no a ibu e he 16:1w5 o he g oup 1 o g oup 2 o G- bac e ia,
mo eo e hey we e loaded up simila ly wi h ungi. Second he VAM a e usually abundan
in soils, because hey o m a symbio ic ela ionship wi h up o 80% o land plan s (Madan
e al., 2002). Thi d 16:1w5 beha ed simila ly wi h ungi in u iliza ion o in es iga ed
LMWOS (especially o ca boxylic acids). All hese ac o s suppo he in e p e a ion ha
16:1w5 e lec VAM in his soil. Fo ensu ing he in e p e a ion o 16:1w5 as VAM a y
acid, he simul aneous analysis o 16:1w5 in PLFAs and neu al lipids should be done. As
a as we didn’ measu e his in ou expe imen , he in e p e a ion o 16:1w5 as VAM is
no assu ed.
2.1.4.3
Inco po a ion o LMWOS in o PLFAs
Amino acids
The obse ed dominan ole o bac e ia in amino acid u ilisa ion is in ag eemen
wi h p e ious s udies, which ound ha he ela i e inco po a ion o
13
C om glu ama e
( he added amoun was 50 µg C g
-1
soil) in o bac e ia was high, whe eas inco po a ion
in o ungi was signi ican ly lowe (B an e al., 2006; Rinnan and Båå h, 2009). Ac inomy-
ce es u ilised amino acids in a o es soil, simila o G+ bac e ia (B an e al., 2006). Ou
esul s wi h ag icul u al soil suppo hose o B an e al. (2006) and con i m he heo y
ha amino acid u no e is mainly con olled by mic obial ac i i y and no by mic obial
communi y s uc u e (Jones e al., 2005).
We demons a ed a p e e ed inco po a ion o alanine han glu ama e in o PLFAs
and a highe eplacemen o PLFA-C by alanine
13
C han glu ama e
13
C (Fig. 2, bo om).
Despi e a simila up ake o alanine and glu ama e in o EMB, hei con as ing inco po a-
ion in o PLFAs shows di e ences in in acellula me abolisa ion: alanine C is di ec ly
con e ed o ace yl-CoA, he di ec p ecu so o a y acids, whe eas complex ene gy-
consuming pa hways a e needed o ans o m glu ama e C in o ace yl-CoA (Fig. 5). In
addi ion, he o ma ion o ace yl-CoA om alanine causes a loss o one- hi d o i s C
backbone compa ed o a h ee- i hs loss o C om glu ama e i con e ed o ace yl-CoA
(Apos el e al., 2013). This also con ibu es o he lowe inco po a ion o glu ama e-C in o
PLFAs. Thus, ou esul s con i m hose o p e ious s udies on me abolic acing, showing
ha in acellula me abolisa ion is he mas e p ocess ha de e mines he a e o amino
acid C in soils (Apos el e al., 2013; Dippold and Kuzyako , 2013; Knowles e al., 2010).
Publica ions and Manusc ip s
64
Suga s
The p e e ence o bac e ia o glucose u iliza ion compa ed o ungi, co esponds
wi h he dominance o bac e ia wi hin he soil mic obial communi y, bu can also be a -
ibu ed o he mo e e icien up ake o LMWOS by bac e ia (Moo e e al., 2005) espe-
cially i low concen a ions o LMWOS a e applied (Reischke e al., 2014). This was e-
ealed p e iously by a highe ela i e glucose inco po a ion in o bac e ia (B an e al.,
2006). The p e e en ial inco po a ion o suc ose in o bac e ial a y acids (16:1
ω
7 and
18:1
ω
7) was also epo ed by No ingham e al. (2009), who no ed he impo ance o he
16:1w7 bioma ke in he con ol o p iming e ec s. Thus, G- bac e ia (co esponding o
ou G-1 g oup) ep esen a g oup whose g ow h is based on easily a ailable subs a es
and which a e he mos compe i i e o LMWOS in many ecosys ems (T eonis e al.,
2004). Hence, he majo i y o s udies show ha bac e ia a e he mos ele an g oup o
he up ake and deg ada ion o easily a ailable subs a es e.g., ollowing he ini ial s age
o li e decomposi ion, whe eas ungi decompose mo e complex subs a es ha emain
a la e s ages (Moo e-Kuce a and Dick, 2008). Howe e , s udies based on nuclea mag-
ne ic esonance showed he signi ican u iliza ion o
13
C om glucose o he o ma ion o
unsa u a ed iacylglyce ols, ypical s o age me aboli es o euka yo es (Lundbe g e al.,
2001). Based on hese esul s, i has been sugges ed ha ungi a e he mos ac i e o -
ganisms in glucose deg ada ion. In e p e a ions in ou s udy a e based on memb ane
lipids – a subs ance class whose s uc u e, unc ion and biosyn he ic pa hways a e simi-
la be ween many p oka yo es and euka yo es. Thus, a compa ison o he u iliza ion pa -
e n is p obably mo e eliable i unc ional and biosyn he ically compa able compounds
a e included (Rinnan and Båå h, 2009).
The up ake pa e n o ibose was ela i ely simila o ha o glucose (Fig. 3, op),
wi h p edominan u iliza ion by G- bac e ia. This p ima y inco po a ion o pen ose by G-
bac e ia was also cha ac e ised by 13C-xylose u iliza ion (Wald op and Fi es one, 2004)
a simila s uc u e and hus p esumably a simila up ake and me abolism.
The high pe -
cen age inco po a ion o ibose
13
C in o EMB compa ed o he ela i ely small amoun o
13
C de ec ed in PLFAs can be explained by he use o ibose o he syn hesis o o he
cell polyme s. A e modi ica ion in he pen ose-phospha e pa hway and phospho yla ion,
ibose is likely o become a subuni o ibonucleo ides and less used o a y acid bio-
syn hesis (Fig. 5). Ribonucleo ides a e ex ac ed a e chlo o o m umiga ion and his can
explain high
13
C inco po a ion in he mic obial pool. Only G-1, he mos ac i e g oup in
LMWOS u iliza ion, inco po a ed high amoun s o ibose in o PLFAs, i.e. pen ose-
phospha e in e media es. This accoun s o high in acellula u no e o his mos ac i e
mic obial g oup.
Publica ions and Manusc ip s
65
Ca boxylic acids
Ace a e is a ubiqui ous subs a e in soil: i is he main p oduc o lipid deg ada ion,
he main subs ance o plan li e anae obic decomposi ion (Rei h e al., 2002), p esen a
high concen a ions in slu y (Laughlin e al., 2009) and is known as di ec p ecu so ole
o he o ma ion o a y acids
.
The amoun o ace a e inco po a ed in o memb anes o G- bac e ia 1 (16:1w7c and
18:1w7c) was 5- old highe han o mos o he o he PLFAs (Fig. 4, op). Simila o he
o he LMWOS, his migh be a esul o hei highe abundance wi hin he mic obial com-
muni y and hei apid up ake o LMWOS. A simila high eco e y o 13C om ace a e in
16:1w7c and 18:1w7c PLFAs was epo ed in expe imen s wi h anoxic b ackish sedimen
(Boschke e al., 2001). Ou expe imen wi h well-ae a ed ag icul u al soil showed ha he
high compe i i eness o hese G- bac e ia o ace a e does no depend on he oxygen
supply. In addi ion, expe imen s wi h sedimen and g oundwa e samples showed ha
only ew gene a we e in ol ed in ace a e deg ada ion (Pombo e al., 2005). The 16:1w7c
PLFA has been sugges ed as a bioma ke o ace a e-oxidising sulpha e- educing bac e-
ia. This anae obic deg ada ion can only occu in O
2
-de icien mic ohabi a s such as ag-
g ega e co es and is e y unlikely o play a ele an ole in eshly illed soil.
Fa y acids ha cha ac e ise G+ bac e ia (such as i15:0, i16:0, i17:0, a17:0) we e
also en iched, bu o a much lowe deg ee han G- bac e ial a y acids (Fig. 4, op). Simi-
la esul s we e ob ained wi h anoxic b ackish sedimen s, whe e 10Me16:0, cy17:0, i15:0
and a15:0 PLFAs we e en iched in
13
C om ace a e and we e ela ed o sulpha e-
educing bac e ia (Boschke e al., 2001). Ca boxylic acids we e he only subs a e class
whe e ungal up ake and inco po a ion could compe e wi h hose o p oka yo ic, G+
g oups. Thus, al hough ungi a e less compe i i e o he mos LMWOS, hey p e e acidic
subs a es wi hin he LMWOS (Rinnan and Båå h, 2009). This co ela es wi h hei p e -
e ence o acid soil condi ions, whe e acidic (non-neu alised), mo e complex subs a es,
domina e (Haide , 1996).
A compa ison o palmi a e and ace a e u iliza ion in soils is impo an because ace-
a e is a di ec mic obial p ecu so o palmi a e syn hesis. The e a e h ee pa hways o
he inco po a ion o palmi a e in o phospholipids: 1) pa ial s ep-by-s ep deg ada ion o
C2-uni s wi hou o al b eakdown o palmi a e can occu and subsequen ly, only pa s o
he molecule a e used o u he biosyn hesis (Rhead e al., 1971); 2) he esyn hesis
pa hway includes he comple e deg ada ion o he molecule o ace yl-CoA and he ollow-
ing syn hesis o new a y acids by a se ies o enzyma ic eac ions (Rhead e al., 1971);
3) he al e na i e is he u iliza ion o palmi a e di ec ly wi hou u he ans o ma ion, be-
cause i is he mos abundan a y acids in mic oo ganisms and migh be sligh ly modi-
ied by elonga ion o desa u a ion. Acco ding o he i s pa hway, palmi a e should ha e a
Publica ions and Manusc ip s
72
Supplemen a y Da a
Supplemen a y Table A1: Fa y acids in he ex e nal s anda d
Supplemen a y Table A1: Resul s o ac o analysis: Fac o loadings and g ouping o a y
acids de i ed om ac o loadings and PLFAs li e a u e.

Publica ions and Manusc ip s
73
Supplemen a y Table A3: Nes ed ANOVA be ween classes o LMWOS and single sub-
s ances nes ed in class o LMWOS o soil, mic obial biomass and PLFAs. Deg ees o
eedom (d ), alues (F) and signi icance le el (p) a e shown o he wo ime poin s.
Publica ions and Manusc ip s
74
2.2 S udy 2: Imp o ed δ
13
C analysis o amino suga s
in soil by Ion Ch oma og aphy – Oxida ion – Iso-
ope Ra io Mass Spec ome y
SHORT TITLE: Amino suga
δ
13
C analysis
by Ion Ch oma og aphy - Iso ope Ra io Mass Spec ome y
Michaela A. Dippold
1,2,3
, S e anie Boesel
2
, Anna Gunina
1,3
, Yako Kuzyako
3,4
,
B uno Glase
2
1
Depa men o Ag oecosys em Resea ch, BayCEER, Uni e si y o Bay eu h
2
Depa men o Soil Biochemis y, Ins i u e o Ag icul u al and Nu i ional Science,
Ma in-Lu he Uni e si y Halle-Wi enbe g
3
Depa men o Ag icul u al Soil Science, Uni e si y o Gö ingen, Ge many
4
Depa men o Soil Science o Tempe a e Ecosys ems, Geo g-Augus Uni e si y o
Gö ingen
Co esponding Au ho :
Michaela Dippold
Depa men o Ag icul u al Soil Science
Geo g-Augus Uni e si y o Goe ingen
Buesgenweg 2
97077 Goe ingen
email: [email p o ec ed]
Tel.: 0921/552187
Fax.: 0921/552246
Publica ions and Manusc ip s
75
Abs ac
Ra ionale: Amino suga s build up mic obial cell walls and a e impo an com-
pounds o soil o ganic ma e . To e alua e hei sou ces and u no e ,
δ
13
C analysis o
soil-de i ed amino suga s by liquid ch oma og aphy was ecen ly sugges ed. Howe e ,
amino suga
δ
13
C de e mina ion emains challenging due o 1) a s ong ma ix e ec , 2)
CO
2
-binding by alkaline eluen s, and 3) s ongly di e en ch oma og aphic beha io and
concen a ions o basic and acidic amino suga s. To o e come hese di icul ies we es-
ablished an ion ch oma og aphy-oxida ion-iso ope a io mass spec ome y me hod o
imp o e and acili a e soil amino suga analysis.
Me hod: A e acid hyd olysis o soil samples, he ex ac was pu i ied om sal s
and addi ional componen s impeding ch oma og aphic esolu ion. Amino suga concen-
a ions and
δ
13
C alues we e analyzed by coupling an ion ch oma og aph o an iso ope
a io mass spec ome e . The accu acy and p ecision o quan i ica ion and
δ
13
C de e mi-
na ion we e assessed.
Resul s: In e nal s anda ds enabled co ec ion o losses du ing analysis, wi h a
ela i e s anda d de ia ion < 6%. The highe magni ude peaks o basic compa ed o
acidic amino suga s equi ed an amoun -dependen co ec ion o
δ
13
C alues. This co -
ec ion allowed o dec ease he accu acy o
δ
13
C de e mina ion o < 1.5‰ and hei p eci-
sion o < 0.5‰ o basic and acidic amino suga s in a single un.
Conclusion: This me hod enables pa allel quan i ica ion and
δ
13
C de e mina ion o
basic and acidic amino suga s in a single ch oma og am due o he ad an ages o cou-
pling an ion ch oma og aph o he iso ope a io mass spec ome e . Small adjus men s o
sample amoun and injec ion olume a e necessa y o op imize p ecision and accu acy
o indi idual soils.
Keywo ds
: compound-speci ic iso ope a io mass spec ome y, amino suga s, soil o -
ganic ma e , mic obial bioma ke analysis, ion ch oma og aphy, IC-O-IRMS coupling.
Publica ions and Manusc ip s
76
2.2.1 In oduc ion
The g ea ele ance o mic obial compounds wi hin soil o ganic ma e (SOM) be-
came e iden wi hin he las decade. Mic obial cell wall compounds seem o be he mos
ele an mic obial-de i ed compound class wi hin slow cycling SOM, as hey 1) a e highly
polyme ic subs ances (Amelung, 2003) and 2) s abilized by in e ac ion wi h soil su aces
(Amelung e al., 2001; Mil ne e al., 2011). Thus, an inc easing in e es a ose o in es i-
ga e hei u no e and accumula ion in soils (Mil ne e al., 2011). Beside i s con ibu ion
o he soil o ganic C (SOC) pool, amino suga s a e - oge he wi h p o eins – he com-
pound classes linking he C and N cycles in soil and con ibu e signi ican ly o he soil
o ganic N (Amelung, 2003). In addi ion, amino suga s p o ide in o ma ion abou he mi-
c obial communi y s uc u e. Bac e ial cell walls consis o pep idoglycan – a polyme o
N-ace ylmu amic acid and N-ace ylglucosamine whe eas ungal cell walls consis o chi-
in, a N-ace ylglucosamine polyme (Engelking e al., 2007; Glase e al., 2004). The o i-
gin o mannosamine and galac osamine, addi ional amino suga s ound in hyd olysis ex-
ac s o soils, a e s ill deba ed.
In con as o cell memb ane compounds like phospholipids, which u n o e apidly
in soils (Re hemeye e al., 2004), amino suga s a e mo e s able. Con ibu ion o li ing
biomass e sus nec omass in soils (Glase e al., 2004) o ungal and bac e ial biomass
(Joe gensen and Wiche n, 2008) as well as eliable and gene ally accep ed esul s on
hei u no e ime in soils a e s ill a e (Amelung e al., 2008; Glase , 2005) as no me h-
ods o
14
C measu emen s o amino suga s, nei he in hei na u al abundance no
14
C-
labeled, ha e been epo ed in he li e a u e o ou knowledge. Recen app oaches ha e
ocused on de e mina ions o
δ
13
C o
δ
15
N alues o amino suga s. These s udies s a ed
om he quan i ica ion o amino suga s by gas ch oma og aphy (Gue an and Moss,
1984; He e al., 2006; Zhang and Amelung, 1996) and con inued wi h gas ch oma og a-
phy-combus ion-iso ope a io mass spec ome y (GC-C-IRMS) (Glase and G oss,
2005). Howe e ,
δ
13
C-de e mina ion by GC-C-IRMS has agg a a ing sho comings
(Decock e al., 2009):
13
C ac iona ion occu s du ing measu emen ; he esul ing o se
and amoun dependence o he iso ope signal can in pa be co ec ed by he use o ex-
e nal s anda d (Glase and Amelung, 2002; Schmi e al., 2003). Howe e , he g ea e
he amoun o in oduced de i a i e C compa ed o C a oms o in e es , he la ge he
e o in he

13
C de e mina ion ha s ill emains a e applying co ec ion unc ions
(Decock e al., 2009; G oss and Glase , 2004).
As amino suga s a e wa e -soluble low molecula weigh o ganic subs ances, hey
can also be quan i ied by high pe o mance liquid ch oma og aphy (HPLC) (Appuhn e
al., 2004; Indo e al., 2011). The e o e, cu en me hodological de elopmen s ha e o-
Publica ions and Manusc ip s
77
cused on he es ablishmen o liquid ch oma og aphy-oxida ion-iso ope a io mass spec-
ome y (LC-O-IRMS) me hods (K ummen e al., 2004) o
δ
13
C measu emen o amino
suga s (Bode e al., 2009), which ha e al eady e ealed i s high po en ial o applica ion
in soil science (Bai e al., 2013; Bode e al., 2013; Indo e al., 2012).
Many LC-O-IRMS me hods and in pa icula he amino suga me hod a e no ou-
inely used. Con en ional liquid ch oma og aphs a e cons uc ed o o ganic eluen s and
p oblems occu i con inuously used wi h s ong acids o bases. Howe e , pe o ming LC-
O-IRMS analysis o
δ
13
C de e mina ion does no allow any o ganic eluen s i.e. o ganic C.
Hence, liquid ch oma og aphy is es ic ed o ion exchange columns which implies he
use o sal solu ions o acids and bases as eluen s (Basle and Dyckmans, in p ess; Bode
e al., 2009). Thus, me allic ions can be dissol ed om s ainless s eel pumps o capilla -
ies and sal c ys alliza ion occu s wi hin he sys em (Bode e al., 2009; Rinne e al.,
2012). This causes a loss in he pe o mance o he columns as well as blockages o he
sys em. To p e en such p oblems, ime and money consuming pu ging s eps ha e o be
implemen ed be ween sample measu emen s (Bode e al., 2009; Rinne e al., 2012). In
addi ion, any con amina ion by HCO
3-
has o be a oided o
δ
13
C de e mina ion as HCO
3-
inc eases C backg ound (i.e. baseline) and i will in luence he
δ
13
C alue o he analy es.
Howe e , liquid ch oma og aphs a e
pe se
no cons uc ed o a oid gas di usion in o he
sys em. Thus, p e-degassing o eluen s ha e o be pe o med o enable ca bona e- ee
ch oma og aphy – especially i bases a e used as eluen s. In addi ion, basic amino sug-
a s (glucosamine, galac osamine and mannosamine) show g ea ly di e en ch oma-
og aphic beha io han he acidic mu amic acid. Thus, a high g adien wi h he eluen s
has o be d i en, leading o s ong elu ion o he ma ix, especially o soils (Bode e al.,
2009). In addi ion, concen a ions o mu amic acid a e en o hund ed imes lowe han
hose o basic amino suga s. This hampe s quan i ica ion due o he limi ed linea ange
o he de ec o s as well as
δ
13
C de e mina ion due o a limi ed ange o peak a ea wi h
ep oducible esul s. The e o e, cu en me hods use a double measu emen wi h di e -
en ch oma og aphies o i s measu e mu amic acid and a e wa ds basic amino suga s
(Bode e al., 2009). This double measu emen as well as he addi ional e o equi ed o
sol en - ee HPLC me hods ende s ou ine measu emen o
δ
13
C alues o amino suga s
nea ly impossible.
The aim o his s udy was o es ablish an ion ch oma og aphy–oxida ion–iso ope a-
io mass spec ome y (IC-O-IRMS) me hod o quan i ica ion and
δ
13
C de e mina ion o
soil-de i ed amino suga s. We hypo hesized ha using an ion ch oma og aph would
s ongly acili a e IRMS measu emen o many bioma ke s, as some basic equi emen s
like ca bona e- ee measu emen o me al- ee sys ems a e al eady ul illed by he in-
s umen . In addi ion, we in ended o op imize amino suga pu i ica ion o educe ca ionic

Publica ions and Manusc ip s
78
con amina ion and ma ix peaks o igina ing om soil. The aim was o p o ide a me hod
enabling a ou ine applica ion o
δ
13
C amino suga measu emen s, which a e c ucial e-
ga ding he inc easing in e es in mic obial con ibu ions o s able SOM.
2.2.2 Ma e ial and Me hods
2.2.2.1
Soil
Topsoil (0-10 cm) om he Ap ho izon o a sil loamy haplic Lu isol (WRB, 2006)
was collec ed om a long- e m cul i a ed ield in Ba a ia (49.907 N, 11.152 E,
501 m. a. s. l, mean annual empe a u e 6-7 °C, mean annual p ecipi a ion 874 mm). The
soil had a pH
KCl
o 4.88 and pH
H2O
o 6.49, TOC and TN con en we e 1.77% and 0.19%,
espec i ely, and po en ial ca ion exchange capaci y was 13.6 cmol
c
kg
-1
. Field esh soil
was sie ed o 2 mm and all oo s we e emo ed wi h weeze s. Soil was hen eeze
d ied, ball milled and 500 mg o he esul ing powde we e used o each hyd olysis.
2.2.2.2
Chemicals, eagen s and ex e nal and in e nal s anda ds
All chemicals o hyd olysis and pu i ica ion we e ob ained om Sigma-Ald ich (S .
Louis, MO, USA) wi h a minimum g ade o “p o analysis” (>99.0% pu i y). Fo ion ch o-
ma og aphy, a 50-52%, ul a-pu e NaOH solu ion was pu chased om Sigma Ald ich (S .
Louis, MO, USA). NaNO
3
-solu ion (0.01 M) was p oduced om me al- ee sodium ni a e,
pu a onic (99.999% pu i y, Al a Aesa , Ka ls uhe, Ge many). Fo oxida ion, a 0.26 M
sodium pe sul a e solu ion and 10% phospho ic acid solu ions we e used (Sigma Ald ich,
S . Louis, MO, USA).
Me hylglucamine p. a. (5 mg mL
-1
) and uc ose p.a. (1 mg mL
-1
) (Sigma Ald ich,
Louis, MO, USA) we e used as he i s and second in e nal s anda ds (IS1 and IS2),
espec i ely. S ock solu ions o ex e nal s anda ds con ained me hylglucamine, gluco-
samine, mannosamine and galac osamine a concen a ions o 5, 14, 1.5 and 20 mg l
-1
(Sigma Ald ich, Louis, MO, USA) and mu amic acid (To on o Resea ch Chemicals Inc.,
To on o, Canada) a 7.5 mg l
-1
. The IAEA-calib a ed
δ
13
C alue o each ex e nal s anda d
was de e mined by epea ed Elemen al Analyze -Iso ope Ra io Mass Spec ome y
(Flash 2000 HT Plus Elemen al Analyze and Del a V Ad an age Iso ope Ra io Mass
Spec ome e , bo h om The mo-Fishe , B emen, Ge many) measu emen o hese sub-
s ances and calib a ed agains ce i ied s anda ds o he In e na ional A omic Ene gy
Agency IAEA (IAEA-CH6: -10.4‰, IAEA-CH7 -31.8‰ and USGS41 37.8‰) e sus Pee
Dee Belemni e (PDB).
Publica ions and Manusc ip s
79
2.2.2.3
Soil hyd olysis and ion emo al
Soil hyd olysis and ion emo al we e pe o med acco ding o Zhang and Amelung
(1996), which was op imized o
δ
13
C de e mina ion by Glase and G oss (2005). B ie ly,
hyd olysis was pe o med wi h 10 mL o 6 M HCl a 105 °C o 8 h. The il a e ex ac
was d ied comple ely and edissol ed in 20 mL H
2
O. One hund ed mic oli e s o he IS1
me hylglucamine (i.e. 50 µg) we e hen added. The pH was adjus ed o 6.6-6.8 wi h 0.6 M
KOH and p ecipi a ed i on was emo ed by cen i uga ion (4000 pm o 15 min). A e
eeze-d ying he esidue was edissol ed in 5 mL o d y me hanol and sal p ecipi a es
we e emo ed by cen i uga ion (4000 pm o 10 min). The supe na an was d ied unde
a gen le s eam o N
2
and s o ed ozen un il column pu i ica ion.
2.2.2.4
Pu i ica ion by ca ion exchange column
Liquid ch oma og aphy equi es a column pu i ica ion o emo e hyd olysable non-
ca ionic compounds like monosaccha ides and ca boxylic acids om he ex ac . A ca ion
exchange column (AG 50W-X8 Resin, H
+
o m, mesh size 100-200, Bio ad, Munich,
Ge many) was used as sugges ed by Indo e al. (2013): a hin laye o clean glass wool
was ins alled unde 4 cm o ca ion exchange esin in he glass column (inne diame e :
0.8 cm). Resin was illed in by insing wi h ~10 mL o 0.1 M HCl solu ion o ensu e he H
+
o m o he so ben , co e ed wi h a hin laye o glass wool and p econdi ioned wi h 5 mL
o wa e . D ied ex ac s we e edissol ed in ~1 mL o wa e wi h one d op o 0.1 M HCl o
ensu e he ca ionic o m o mu amic acid. A e ans e ing he sample on o he column,
neu al and anionic compounds we e elu ed wi h 8 mL wa e . The ca ionic ac ion con-
aining he amino suga s was elu ed by 15 mL 0.5 M HCl, eeze-d ied and ans e ed
wi h 5 mL o d y me hanol. A e e apo a ion o he me hanol by a gen le s eam o d ied
N
2
, he sample can be s o ed ozen (-20 °C) o a leas one mon h. Fo subsequen
measu emen , he samples we e e-dissol ed in 200 µL wa e wi h he addi ion o 50 µL
o IS2 solu ion and measu ed wi hin 24 hou s a e e-dissol ing.
2.2.2.5
De elopmen o he measu emen by IC-O-IRMS
All measu emen s we e pe o med by a Dionex ICS-5000 SP ion ch oma og aphy
sys em coupled by an LC IsoLink o a Del a V Ad an age Iso ope Ra io Mass Spec ome-
e (Supplemen a y Figu e 1) (all componen s om The mo-Fishe , B emen, Ge many).
Ch oma og aphic condi ions we e modi ied and op imized wi h he aim o eaching base-
line sepa a ion and a esolu ion ac o Rs g ea e han 1.
Publica ions and Manusc ip s
80
( )
12
12
ww5.0
Rs +⋅
−
=
equa ion 1
1
and
2
a e he e en ion imes o wo neighbo ing peaks and w ep esen s hei e-
spec i e peak wid h a he angen s baseline (Figu e 1).
Nine mic oli e s o he wa e -dissol ed sample o ex e nal s anda d we e injec ed
ia a 25 µL injec ion loop and he injec ion ime was de ined as 0 sec. Ch oma og aphy
was pe o med by a Ca boPac
TM
PA 20 analy ical anion exchange column (3 x 150 mm,
6.5 µm) which was p eceded by a PA 20 gua d column (Bode e al., 2009) (bo h om
Dionex, Ams e dam, The Ne he lands). The elu ion sequence con ained a p econdi ion-
ing be o e injec ion (15 min wi h 200 mM NaOH and 10 min wi h 8 mM NaOH). Elu ion
sequence las ed o 35 min in o al and was pe o med a cons an empe a u e o 30 °C
and a low a e o 0.4 ml min
-1
. 8 mM NaOH was inc eased a e 11 min o 8 mM NaOH
wi h a pulse o 2.5 mM NaNO
3
un il 15
h
minu e. Then NaNO
3
was dec eased and NaOH
concen a ion inc eased o inal 20 min o ch oma og am (de ails in Supplemen a y Ta-
ble 1).
We measu ed ex e nal s anda ds a ou concen a ions (e.g. 50, 100, 175 and
250 µL o he s ock solu ion) a leas once be o e and once a e a sample ba ch. A sam-
ple ba ch consis ed o 4–6 samples, each measu ed 4 imes. A sample ba ch was always
measu ed once in i s en i e y and hen epea ed h ee imes.
In eg a ion was pe o med by Isoda 3.0 (The mo Fishe Scien i ic, B emen, Ge -
many) wi h he ollowing pa ame e s: s a slope 1 mV/s, end slope 2 mV/s, peak min
50 mV, peak esolu ion 50% and an indi idual backg ound.
Publica ions and Manusc ip s
81
Fig. 1 Ch oma og am o ex e nal s anda d ( op) and un-spiked sample (down). Fi s
and second in e nal s anda ds as well as basic amino suga s (galac osamine,
mannosamine and glucosamine) and acidic mu amic acid a e ma ked. Peak
esolu ion Rs is included o he iple o basic amino suga s in he uppe
ch oma og am o he ex e nal s anda d and Rs o mu amic acid and i s p e-
ceeding ma ix peak is shown in he ch oma og am o he sample.
2.2.2.6
E alua ion o amino suga quan i ica ion ia IC-O-IRMS
To alida e he me hod by s anda d addi ion, he s anda d mix u e se ing as ex e -
nal s anda ds was added o he hyd olysis ex ac s. The amoun s o subs ance added
we e in he ange o 0, 1.3, 1.7, 2.1 and 3 imes o he expec ed concen a ions.
The da a om he s anda d addi ion expe imen we e s a is ically e alua ed acco d-
ing o Bi k e al. (2012): Fo each subs ance (including IS1) a linea eg ession was i ed
by he me hod o leas squa es o he measu ed amoun s as a unc ion o he added
amoun s pe sample (Supplemen a y Figu e 2). The y-in e cep ep esen ed he i ed
amoun o subs ance in soil and he slope ga e he mean eco e y o a subs ance. The
signi icance o eg ession was es ed and S e en’s Runs Tes was pe o med o iden i y
de ia ions om linea i y. Signi ican di e ences be ween eco e ies we e de ec ed by
co a iance analysis (ANCOVA) o he slopes. All eg ession pa ame e s we e calcula ed
Publica ions and Manusc ip s
88
LoQ wi hou ha ing an o e load in he glucosamine peak. Unde such special condi ions
a double measu emen wi h high concen a ed sample o de e mina ion o mu amic acid
δ
13
C alues and dilu ed concen a ion o de e mina ion o glucosamine
δ
13
C alues migh
be necessa y.
A e age amino suga
δ
13
C alues di e o ~0.1 o 1.1‰ wi hin he basic (Bode e
al., 2009; Bode e al., 2013) and o mo e han 3-5‰ be ween he basic and acidic amino
suga s (Bode e al., 2009; Bode e al., 2013; Glase , 1999) and di e o a ound 7‰ om
bulk SOC (Glase and G oss, 2005). The achie ed accu acies o indi idual amino suga s
enable o dis inguish amino suga om hei C sou ces e en unde na u al abundance
condi ions. Resul ing p ecisions (0.5‰) a e lowe han di e ences be ween basic and
acidic amino suga s and consequen ly enables o iden i y mic obial g oup speci ics in
amino suga o ma ion (e.g. speci ics in he used subs a es o he ac iona ions in bio-
chemical o ma ion pa hways). Especially in expe imen s leading o a highe
δ
13
C di e -
ences in amino suga s like C3 o C4 C sou ce changes (Indo e al., 2012), FACE ex-
pe imen s (Glase and G oss, 2005) o applica ion o labeled subs a es (Bode e al.,
2013) his me hod can ully dis inguish C sou ces and indi iduali ies in cell wall o ma ion
o ungi and bac e ia.
In summa y, his me hod enables a combined de e mina ion o
δ
13
C alues o
amino suga s o he majo i y o soils. Howe e , adjus men s o new sample ypes a e
necessa y o iden i y he op imum amoun o sample o hyd olyze o he inal olume o
injec so ha he op imum ange o accu acy and p ecision o he
δ
13
C alues a e me .
Fig. 3 Amoun -dependen unc ion o es ima ion o s anda d e o o δ
13
C (δ
inal
(Ai)
de e mina ion calcula ed acco ding o equa ion 6.

Publica ions and Manusc ip s
89
2.2.3.5
Ad an ages o IC-O-IRMS
Many p e ious s udies epo ed se e e p oblems wi h LC-O-IRMS, e.g. he impos-
sibili y o measu ing mu amic acid in non-spiked samples due o e y low peak a eas o
he equi emen o ime-consuming pu ging s eps o main ain pe o mance o he PA 20
column (Bode e al., 2009). The absence o hese issues in he cu en ly p oposed
me hod can mainly be a ibu ed o he ad an ages o IC o e HPLC. Ion ch oma og aphs
a e ee o me als: all elemen s ha a e in con ac wi h sample o eluen s a e made om
polye he e he ke on (peek). Thus, me al con amina ion can o igina e only om he sam-
ple. Howe e , ou me hod con ains i on and sal p ecipi a ion s eps, emo ing all (po en-
ially column des oying) ca ions. This no only educes measu emen ime bu also e-
duces cos s as, e.g., in-line high p essu e il e s p o ec ing he column om colloids and
me al ions a e no needed. E en a e 600 injec ions, no dec ease in pe o mance o he
PA 20 column was de ec ed and he p e-column did no need o be exchanged.
In addi ion, he CO
2
- igh cons uc ion o Ion Ch oma og aphs is a g ea ad an age
o
δ
13
C de e mina ion as no shi s in he
δ
13
C alue due o inc easing ca bona e back-
g ound occu ed. The e o e, e en CO
2
-binding eluen s, like NaOH, do no cause p ob-
lems o ch oma og aphy and iso ope a io mass spec ome y. In addi ion, Ion Ch o-
ma og aphs a e ou inely equipped wi h a degasse , which keeps he eluen s and oxidiz-
ing eagen s o he Isolink CO
2
- ee. Thus, al hough acquisi ion cos s may be highe , he
imp o ed pe o mance, highe sample h oughpu and lowe ollow-up cos s e lec he
clea ad an ages o ion ch oma og aphs o imp o ing LC-O-IRMS.
2.2.4 Conclusions
Amino suga s a e impo an bioma ke s o esea ch on bac e ial and ungal con i-
bu ion o SOM. This new me hod enables pa allel quan i ica ion and
δ
13
C de e mina ion
o he mos equen amino suga s in soils and hus se s he p econdi ions o wide adop-
ion o
δ
13
C amino suga de e mina ion in soil science.
The combina ion o i on and sal emo al om gas ch oma og aphy p o ocols wi h
pu i ica ion ia ca ion exchange esins adap ed om liquid ch oma og aphy me hods
p o ed o be an op imal sample p epa a ion o ion ch oma og aphy including ch oma-
og aphic sepa a ion, sys em s abili y and longe i y o sys em componen s. In addi ion,
using ion ch oma og aph se s clea ad an ages o e HPLCs as me al and ca bona e
exclusion om he sys em a oids column con amina ion as well as dis u bance o
δ
13
C
de e mina ion by a ca bona e backg ound.
Publica ions and Manusc ip s
90
These imp o emen s o e p e ious me hods enabled pa allel quan i ica ion and
δ
13
C de e mina ion o high-concen a ed basic amino suga s and low-concen a ed mu-
amic acid. Reco e ies anged om 57 o 66% and could be co ec ed by using me hyl-
glucamine as he i s in e nal s anda d. The quan i ica ion limi o mu amic acid, he
compound wi h he lowes concen a ion, was a ound 0.05 mg pe ial o quan i ica ion
and o iso ope measu emen . When mu amic acid exceeded hese alues, glucosamine,
he mos concen a ed compound, was s ill in a linea ange o quan i ica ion and
δ
13
C
measu emen . The accu acy o IC-O-IRMS was be e han 1‰ o basic amino suga s
and be e han 1.5‰ o mu amic acid compa ed o calib a ed EA-IRMS alues. P eci-
sion was amoun -dependen and less han 0.5‰ o e a compa a i ely b oad ange o
a eas. Howe e , he dependence on he ma ix and he a io o mu amic acid o gluco-
samine in indi idual samples necessi a es adjus men in soil amoun o injec ion olume
o achie e he op imal accu acy and p ecision o
δ
13
C.
The quali y o he quan i ica ion and
δ
13
C de e mina ion as well as sample h ough-
pu o his me hod should enable his me hod o be used ou inly in soil science. The ad-
an ages o IC-O-IRMS compa ed o HPLC-O-IRMS a e e iden and migh also b ing
ad an ages o analysis o o he bioma ke s.
Acknowledgmen s
We hank he DFG o inancing he IC-O-IRMS ins umen and he p ojec DFG KU
1184 19/1.
Publica ions and Manusc ip s
91
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ma ke wi h s able iso ope analyses o assessing he ans o ma ion and u no e o
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acids in soil: Assessmen by posi ion-speci ic labeling and C-13-PLFA analysis. Soil
Biology & Biochemis y 67, 31-40.
Appuhn, A., Joe gensen, R.G., Raubuch, M., Schelle , E., Wilke, B., 2004. The au o-
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gas ch oma og aphy-mass spec ome y. Jou nal o Ch oma og aphy A 1242, 1-10.
Bode, S., Dene , K., Boeckx, P., 2009. De elopmen and e alua ion o a high-
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Bode, S., Fancy, R., Boeckx, P., 2013. S able iso ope p obing o amino suga s - a p om-
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Publica ions and Manusc ip s
93
Supplemen a y Da a
Figu e Supplemen a y A1: Scheme o he ins umen coupling: Ion Ch oma og aph is
shown on he le side wi h pump, au osample and de ec o -ch oma og aphy compa -
men . Connec ion o isolink occu s ia a peek capilla y wi h in e posed colloid il e .
Scheme o LC Isolink is adap ed om K ummen e al. (2004).
Figu e Supplemen a y A2: S anda d addi ion line o he quan i ied amino suga s: quan i-
ied amoun pe g soil is plo ed agains he amoun o spiked amino suga . Slope ep e-
sen s eco e y o he indi idual analy es and y-axis gap ep esen s soil con en wi hou
eco e y co ec ion. Reg ession pa ame e s a e shown in Table 2.

Publica ions and Manusc ip s
94
Supplemen a y Figu e A3: measu ed
δ
13
C alues o spiked samples a e plo ed agains
he pe cen o peak a ea, which is de i ed om he added s anda d: y-in e cep o he
i ed linea eg ession e lec s he i ed alue o soil whe eas
δ
13
C- alue a 100% s an-
da d e lec s he
δ
13
C alue o he added s anda d subs ance
Figu e Supplemen a y A4: a ea-dependan e o e ms o equa ion 6: le side shows he
s anda d e o o he measu emen epe i ion o soil samples and igh side shows he
a ea-dependan e o o he calib a ion/co ec ion unc ion om he ex e nal s anda d line
Publica ions and Manusc ip s
95
Supplemen a y Table A1: Sol en g adien and low condi ions o he IC-O-IRMS sys em
ime 20 mM NaOH 200 mM Na-
OH H
2
O 0.01 M
NaNO
3
low
(ml min
-1
)
-25 min 0% 100% 0% 0% 0.400
-10 min 8% 0% 92% 0% 0.325
11 min 40% 0% 35% 25% 0.400
15 min 45% 0% 45% 10% 0.400
18 min 25% 25% 50% 0% 0.380
35 min s andby
Publica ions and Manusc ip s
96
2.3 S udy 3: Biochemical pa hways o amino acids in
soil: E alua ion by posi ion-speci ic labeling and
13
C-PLFA analysis
Ca olin Apos el
1
, Michaela Dippold
2,3
, B uno Glase
4
, Yako Kuzyako
1,2
1
Depa men o Soil Science o Tempe a e and Bo eal Ecosys ems, Geo g-Augus -
Uni e si y Gö ingen
2
Depa men o Ag icul u al Soil Science, Geo g-Augus -Uni e si y Gö ingen
3
Depa men o Ag oecosys em Resea ch, BayCEER, Uni e si y o Bay eu h
4
Depa men o Soil Biogeochemis y, Ins i u e o Ag icul u al and Nu i ional Science,
Ma in-Lu he -Uni e si y Halle-Wi enbe g
Co esponding Au ho :
Ca olin Apos el
Depa men o Ag oecosys em Resea ch
Uni e si y o Bay eu h
Uni e si ae ss asse 30
95447 Bay eu h
email: [email p o ec ed]
Tel.: 0921/552187
Fax.: 0921/552246
Publica ions and Manusc ip s
97
Abs ac
Mic obial u iliza ion is a key ans o ma ion p ocess o soil o ganic ma e (SOM).
Fo he i s ime, posi ion-speci ic
13
C labeling was combined wi h compound-speci ic
13
C-PLFA analysis o ace me aboli es o wo amino acids in mic obial g oups and o
econs uc de ailed biochemical pa hways. Sho - e m ans o ma ion was assessed by
applying posi ion-speci ically
13
C labeled alanine and glu ama e o soil in a ield expe i-
men . Mic obial u iliza ion o he amino acids’ unc ional g oups was quan i ied by
13
C
inco po a ion in o al mic obial biomass and in dis inc mic obial g oups classi ied by
13
C-
PLFA.
Loss om PLFAs was as es o he highly oxidized ca boxyl g oup o bo h amino
acids, whe eas he educed C posi ions, e.g. C
3-5
, we e p e e en ially inco po a ed in o
mic oo ganisms and hei PLFAs. The inco po a ion o C om alanines’ C
2
posi ion in o
he cell memb ane o g am nega i e bac e ia was highe by mo e han one o de o mag-
ni ude han in o all o he mic obial g oups. Whe eas C
2
o alanine was s ill bound o C
3
a
day 3, he C
2
and C
3
posi ions we e pa ially spli a day 10. In con as , he C
2
o glu a-
ma e was los as e om PLFAs o all mic obial g oups. The di e gence index, which
e lec s ela i e inco po a ion o one posi ion o he inco po a ion o C om all posi ions in
a molecule, e ealed ha disc imina ion be ween posi ions is highes in he ini ial eac-
ions and dec eases wi h ime.
Recons uc ion o mic obial ans o ma ion pa hways showed ha he C
2
posi ion o
alanine is los as e han i s C
3
posi ion ega dless o whe he he molecule is used ana-
o ca abolically. Glu ama e C
2
is inco po a ed in o PLFAs only by wo ou o eigh mic o-
bial g oups ( ungi and pa o g am posi i e p oka yo es). I s inco po a ion in PLFA can
only be explained by ei he he u iliza ion o he glyoxola e bypass o he ans o ma ion
o glu ama e in o aspa a e p io o being ed in o he ci ic acid cycle. Du ing hese pa h-
ways, no C is los as CO
2
bu nei he is ene gy p oduced, making hem ypical C de i-
ciency pa hways. Glu ama e is he e o e a p omising me abolic ace in ega d o eco-
physiology o cells and he e o e changing en i onmen al condi ions.
Analyzing he a e o indi idual C a oms by posi ion-speci ic labeling allows insigh
in o he mechanisms and kine ics o mic obial u iliza ion by a ious mic obial g oups. This
app oach will s ongly imp o e ou unde s anding o soil C luxes.
Key wo ds: me aboli e acing, ans o ma ion pa hways, s able iso ope applica-
ions, mic obial communi y s uc u e and unc ions, compound-speci ic iso ope analysis
Publica ions and Manusc ip s
104
To co ec o amoun -dependen
13
C iso opic ac iona ion du ing measu emen s
(Schmi e al., 2003) and o he addi ion o C du ing de i a iza ion, linea and loga i hmic
eg essions o he ex e nal s anda ds
δ
13
C- alues o hei a ea we e calcula ed. I bo h
eg essions we e signi ican , ha wi h he highe signi icance was applied. As he
δ
13
C-
alue o he de i a iza ing agen s was unknown, he co ec ion was pe o med acco ding
o Glase and Amelung (2002a) (Eq. 5).
%)())(%)((
)(
)(
%)(
ln/ln/
a C Ama C
CN
CN
a C
FSEAlinFAMElinDKFAME
FS
FAME
FS −−
++⋅−⋅=
(5)
wi h: C
FS
(a %) co ec ed
13
C amoun o he a y acid [a %]
C
FAME
(a %) d i -co ec ed
13
C amoun o he FAME [a %]
m
lin/ln
slope o linea /loga i hmic eg ession [a %
·
Vs
-1
]
lin/ln
y-in e cep o linea /loga i hmic eg ession [a %]
A
FAME
a ea o FAME [Vs]
N(C)
FAME
numbe o C a oms in FAME
N(C)
FS
numbe o C a oms in a y acid
C
EA-FS
(a %) measu ed
13
C- alue o a y acid [a %]
2.3.2.3
Di e gence Index
Disc imina ion o C om indi idual posi ions in one molecule du ing up ake and/o
u iliza ion was assessed. The ex en o disc imina ion be ween pools, mic obial g oups
and a wo sampling imes was compa ed as well. Fo bo h o hese asks, he di e ences
in absolu e up ake in o C pools o mic obial g oups had o be ela i ized. The e o e, he
di e gence index (DI) was de ined:
∑
⋅
=
n
i
i
i
C
Cn
DI
1
(6)
wi h: n numbe o C a oms in molecule
C
i
ela i e inco po a ion o ace C [mol
·
mol
-1
]
As equi ed, he DI can be calcula ed wi h ela i e inco po a ion o ace pe bulk
soil, mic obial biomass, single PLFA o
Σ
PLFA o mic obial g oups. The DI compa es he
calcula ed ac ual inco po a ion o C om each posi ion wi h he mean C inco po a ion
om all posi ions. This can be unde s ood as he esul he expe imen s would ha e had
i uni o mly labeled ace s had been used. A DI o 1 would indica e no disc imina ion

Publica ions and Manusc ip s
105
be ween he posi ions; alues abo e 1 indica e p e e en ial inco po a ion, alues below 1
show p e e en ial deg ada ion.
2.3.2.4
S a is ical analysis
Fo he epe i i e measu emen s o d13C- alues, a Nalomo ou lie es wi h signi i-
cance le els o 95% (when ou epe i ions we e a ailable) o 99% (when h ee epe i-
ions we e a ailable) was pe o med. PLFAs we e classi ied in o co esponding mic obial
g oups by a ac o analysis o C con en s o he en i e da ase . Fa y acids wi h a loading
o mo e han 0.5 (absolu e alue) on he same ac o we e ca ego ized wi h ega d o
p e ious s udies (Zelles, 1999; Zelles e al., 1995). All he da a p esen ed in his s udy
we e es ed wi h a one-way analysis o a iance (ANOVA); signi icances we e de e -
mined wi h he Tukey Hones Signi icance Di e ence (Tukey HSD) pos -hoc es wi h a
signi icance le el o 99.5%. All posi ions we e es ed o signi ican di e ences be ween
eco e ies in soil, mic obial biomass and PLFAs. Fo e e y mic obial g oup and soil pool,
he di e ence in DI o he six posi ion-speci ically labeled posi ions was also es ed o
signi icance. All s a is ical es s we e accomplished wi h R e sion 2.9.0 (17.04.2009).
2.3.3 Resul s
2.3.3.1
Inco po a ion o uni o mly labeled amino acids
The C con en in he soil was 1230 µmol
·
g
-1
(Table 2), which co esponds o
15.0 mg C
·
g
-1
soil. O his C, 3.5% is con ained in mic obial biomass, and 0.01% in he
sum o PLFA (
Σ
-PLFA). The inco po a ion o uni o mly
13
C-labeled alanine and glu amic
acid in o soil and mic obial biomass dec eased be ween days 3 and 10. The eco e y in
Σ
-PLFA emained s able o e en inc eased. Reco e y
o applied alanine and glu amic
acid C in soil dec eased by abou hal be ween days 3 and 10. Reco e y om applied
glu amic acid in mic obial biomass dec eased by nea ly 90% be ween days 3 and 10
(Table 2).
Table 2 To al C con en and
13
C inco po a ion o uni o mly labeled amino acids in o soil,
mic obial biomass and sum o PLFA (
Σ
-PLFA).
Publica ions and Manusc ip s
106
2.3.3.2
Inco po a ion o posi ion-speci ically labeled amino acids
Wi h he ool o posi ion-speci ic labeling, we we e able o ace C om indi idual
posi ions o alanine and glu amic acid in o di e en soil C pools. On day 3 (Fig. 1, op), a
clea disc imina ion agains he ca boxyl C o bo h amino acids and glu amic acids amino-
bound posi ion in soil, mic obial biomass and
Σ
-PLFA is e iden . On day 10 (Fig. 1, bo -
om), he eco e y o he ca boxyl C in soil emained s able, while he eco e y o all
o he posi ions in soil dec eased by up o 60% o applied
13
C. This esul s in an equal
eco e y o all posi ions in soil on day 10. In mic obial biomass and
Σ
-PLFA he eco e y
o
13
C om bo h ca boxyl g oups and glu amic acids amino bound posi ion on day 10
was s ill lowe han he
13
C eco e y om o he posi ions o bo h amino acids. In mic obial
biomass, he
13
C eco e y o glu amic acids amino-bound posi ion dec eased by 35%.
The same amoun o
13
C om glu amic acid’s posi ions was eco e ed in
Σ
-PLFA
on bo h
days. Fig. 1 shows ha on day 10, all C om he C
2
and C
3
posi ions o alanine in soil
was loca ed in he mic obial biomass. The ca boxyl C om alanine and glu amic acid,
howe e , was s abilized in soil by o he mechanisms.
Fig. 1 Reco e y o posi ion-speci ically
13
C labeled Ala and Glu in soil, mic obial bio-
mass and
Σ
-PLFA, 3 ( op) and 10 days (bo om) a e applica ion. Le e s indi-
ca e signi ican di e ences (p < 0.05) be ween eco e y bulk soil (a), mic obial
biomass (a’) and
Σ
-PLFA (a’’)
Publica ions and Manusc ip s
107
To iden i y mic obial g oups, a PCA was pe o med on he PLFAs C-con en om
bo h sampling imes. By compa ing classi ica ion in he li e a u e (Z
ELLES
1999; Z
ELLES
e
al.
1995), he a y acid g oups we e ma ched o mic obial g oups and h ough ac o load-
ings hey we e u he subdi ided (Supplemen a y Table 3). Reco e y o applied posi ion-
speci ically labeled C om he wo amino acids in mos mic obial g oups (Fig. 2) shows
he same pa e n as eco e y o applied C in
Σ
-PLFA: The
13
C eco e y om he ca boxyl
g oups is less han 0.1% o
13
C inpu o bo h amino acids on bo h days. On day 3, he
eco e ies o he amino-bound and he me hyl C om alanine we e simila . This pa e n
was di e en on day 10, when he eco e y o alanines amino-bound C was lowe han
ha o i s me hyl g oup. In he i s days a e being aken up by mic oo ganisms and u il-
ized in he cell memb ane, only he C
1
posi ion was spli om he alanine molecule, while
he C
2
and C
3
posi ions we e u ilized oge he .
Fig. 2 Reco e y o applied
13
C om posi ions o alanine ( op) and glu amic acid (bo -
om) in mic obial g oups a e 3 and 10 days. Le e s indica e signi ican di e -
ences (p < 0.05) be ween ca boxyl C (a), amino-bound C (a’) and me hyl C o
alanine o he esidual molecule o glu amic acid (a’’).
Publica ions and Manusc ip s
108
The
13
C eco e y o glu amic acid’s posi ions e eals ha i is ans o med di e -
en ly han alanine. F om bo h he amino-bound and he ca boxyl C, less han 0.4% we e
eco e ed in
Σ
-PLFA on bo h days. In con as , nea ly 4% o he esidual amino acid C
we e eco e ed in
Σ
-PLFA. In he mic obial pa hways bo h C
1
and he C
2
om glu amic
acid we e spli om he esidual molecule, which was hen inco po a ed in o PLFAs.
The maximum inco po a ion o C in o PLFAs om all posi ions o bo h amino acids
was achie ed by he g oup o g am nega i e I (18:1
ω
7c, 18:1
ω
9c) (Fig. 2). This g oup o
g am nega i e p oka yo es ook up 4.5 - 5.5% o he me hyl C
om alanine and also o
he esidual molecules C
om glu amic acid. No o he mic obial g oup ook up mo e han
2% om any posi ion. Mos p oka yo ic g oups inco po a ed mo e C om C
2
and C
3
posi-
ions o alanine han he anae obic bac e ia (cy17:0) and he wo euka yo ic g oups
(Fungi (20:1
ω
9c, 18:2
ω
6,9) and VA-Myco hiza (16:1
ω
5c)).
2.3.3.3
Di e gence Index
The di e gence index (DI) was used o compa e he ex en o inco po a ional dis-
c imina ion o C om di e en posi ions be ween he pools (Fig. 3) and mic obial g oups
(Fig. 4).The DI ela i izes di e ences in absolu e up ake. Rega ding he DI in soil, mic o-
bial biomass and
Σ
-PLFA (Fig. 3), di e ences be ween days 3 and 10 a e ace appli-
ca ion can be obse ed (Fig. 3). The ela i e inco po a ion in soil on day 3 shows a clea
disc imina ion agains he ca boxyl posi ions; on day 10, he e is no signi ican di e ence
in DI be ween any posi ion o alanine o glu amic acid. Al hough only he declined dis-
c imina ion agains alanines ca boxyl C be ween day 3 and 10 is signi ican (p < 0.05),
he educed disc imina ion be ween all posi ions in soil be ween day 3 and day 10 shows
ha du ing he ini ial eac ions, he pa hways o C om di e en posi ions o he wo
amino acids di e g ea ly. Howe e , en days a e applica ion, he sou ce posi ion in he
molecules is no de e mining o ixa ion in soil. In mic obial biomass, a educ ion o dis-
c imina ion be ween posi ions may be aking place – he disc imina ion be ween posi ions
is no signi ican anymo e – bu high s anda d e o s p e en any ce ain conclusions. In
Σ
-PLFA, alanine’s amino-bound posi ion had a DI equal o ha o i s me hyl posi ion on
day 3 bu a lowe DI on day 10. The inco po a ion pa e n o glu amic acids posi ions in o
Σ
-PLFA did no change be ween day 3 and day 10.
Publica ions and Manusc ip s
109
Fig. 3 Di e gence index (DI) e lec ing inco po a ional disc imina ion be ween C posi-
ions in o soil, mic obial biomass and
Σ
-PLFA, 3 (le ) and 10 ( igh ) days a e
applying
13
C-labeled alanine (Ala) and glu amic acid (Glu). Le e s indica e sig-
ni ican di e ences (p < 0.05) in he ela i e inco po a ion o he C posi ions in o
soil (a), mic obial biomass (a’),
Σ
-PLFA (a’’) on day 3, and in o
Σ
-PLFA (a**) on
day 10 a e ace applica ion.
Despi e he di e ences in absolu e
13
C eco e y in PLFAs be ween he mic obial
g oups (Fig. 2), he DI ( ela i e
13
C eco e y) o mos mic obial g oups PLFAs (Fig. 4)
shows a simila pa e n, which also gene ally e lec s he pa e n desc ibed by
Σ
-PLFA
(Fig. 3). In mos PLFAs, he e was an a e age o abo e-a e age ela i e inco po a ion o
he amino-bound g oup o alanine on day 3, which is p ominen on day 10 (Fig. 4). The DI
o he me hyl posi ion o alanine and he esidual molecule o glu amic acid was abo e-
a e age in all mic obial g oups and on bo h days. The amino-bound C o glu amic acid
was inco po a ed less han a e age in all mic obial g oups and on bo h days. Excep ions
o his pa e n we e he g oups o g am posi i e II (i15:0, i17:0) and ungi (20:1
ω
9c,
18:2
ω
6,9), which bo h showed no signi ican disc imina ion agains any posi ion on ei he
days.

Publica ions and Manusc ip s
110
Fig. 4 Di e gence Index (DI), e lec ing disc imina ion be ween C posi ions o alanine
(Ala) and glu amic acid (Glu), 3 ( op) and 10 (bo om) days a e applica ion. Le -
e s indica e signi ican di e ences (p < 0.05) be ween he ela i e inco po a ion
o he C posi ions in o he mic obial g oup a: g am nega i e I, a’: g am nega i e
II, a’’: g am posi i e I, a*: ac inomyce es, a°: VA-myco hiza.
2.3.4 Discussion
2.3.4.1
Inco po a ion o ca bon om amino acids in soil and mic obial bio-
mass
On day 3, he
13
C eco e y om alanine and glu amic acid in soil, mic obial bio-
mass and
Σ
-PLFA shows he same pa e n (Fig. 3). C om he ca boxyl g oup is eco -
e ed less han ha om he amino-bound and me hyl posi ions and he esidual molecule
o glu amic acid in all pools. This was expec ed as he ca boxyl C has he highes oxida-
ion s a e and is he e o e mos p one o being emo ed om he molecules. This is
achie ed by deca boxyla ion. Enzymes necessa y o deca boxyla ion o amino acids
ha e been ound in soil (B aun e al., 1992; Tena e al., 1986) as well as in p oka yo ic
Publica ions and Manusc ip s
111
and euka yo ic mic oo ganisms (Caspi e al., 2008). On day 10, he amoun o ca boxyl C
om bo h amino acids in soil emained s able, while he eco e y o all o he posi ions
dec eased. Al hough i seems con adic o y his can also be explained by he high eac-
i i y o he ca boxyl C: no only can i be oxidized o CO
2
easily, bu i can also eac wi h
o he soil componen s and be hus s abilized. This possible s abiliza ion mechanism is
suppo ed by esul s o Kuzyako (1997), who ound posi ion-speci ically labeled
14
C om
he ca boxyl posi ion o alanine in humic and ul ic acids. As complex mac omolecules,
humic and ul ic acids con ain a a ie y o unc ional g oups such as hyd oxyl g oups,
me hylenes, e he s and es e s in alipha ic chains (Simpson e al., 2002). I is possible o
ca boxyl C om mic obial sou ces o eac wi h humic mac omolecules, e.g. by o ming
es e -linkages wi h hyd oxy g oups. Es e s a e highly ine , he e o e he o me ca boxyl
C will be s abilized om u he deg ada ion.
In con as o ca boxyl C, he
13
C eco e y om he amino-bound and me hyl g oup
o alanine in soil dec eased by up o 60% be ween days 3 and 10. Compa ed o he de-
c ease in eco e y o hese posi ions in soil, he amoun inco po a ed in o mic obial bio-
mass is s ill high on day 10. In mic oo ganisms, alanine can be used ca abolically, in he
ci ic acid cycle and anabolically, e.g. o p oduce suga s o a y acids (Fig. 5) (Caspi e
al., 2008). The i s eac ions o bo h pa hways a e he same. Alanine is i s deamina ed
and hen deca boxyla ed, he ea e he esul ing ace yl eac s wi h coenzyme A o o m
ace yl-CoA. The ace yl-CoA, which consis s o he o me amino-bound and me hyl C
om alanine, is hen ed in o he ci ic acid cycle o used o biosyn hesis. This explains
why C om hose wo posi ions is eco e ed in PLFAs, bu C om he ca boxyl g oup is
no . A e inco po a ion in o PLFAs, C om he o me amino-bound posi ion is on he
e minal posi ion and hus mos p one o being oxidized and deca boxyla ed (Caspi e al.,
2008).This p ocess is hin ed a by he sligh dec ease in ela i e inco po a ion o alanines
amino-bound posi ion be ween day 3 and 10. The inco po a ion o he C om he me hyl
posi ion o alanine and om he esidual molecule o glu amic acid in
Σ
-PLFAs is s ill high
on day 10.
2.3.4.2
Inco po a ion o ace in o he mic obial g oups
The
13
C inco po a ion in o PLFAs o mic obial g oups di e ed by mo e han one o -
de o magni ude (Fig. 2). As hypo hesized, he highes inco po a ion, wi h mo e han 5%
13
C up ake, was eco ded o a g oup o g am nega i e p oka yo es (g am nega i e I).
This i s well wi h he obse a ions by G i i hs e al. (1999) ha g am nega i es eac
as es o addi ion o LMWOS, which gi es hem a compe i i e ad an age.
Publica ions and Manusc ip s
112
Fig. 5 Mic obial ans o ma ion pa hways o alanine (a) and glu amic acid (b, c, d). As
he e a e di e en ans o ma ion pa hways o glu amic acid, i is p esen ed in 3
sub igu es. The en ance o alanine (a) occu s om he bo om (in con as o
glu amic acid, b, c, d,) o he ci ic acid cycle because o i s ini ial ans o ma ion
o ace yl-CoA.
Th ee o he p oka yo ic g oups (g am nega i es II, ac inomyce es and g am posi-
i e II), also achie ed mode a e
13
C inco po a ion. The wo euka yo ic g oups – ungi and
VA-myco hiza – we e unable o ake up as much o he applied amino acid C as he p o-
ka yo ic g oup. This is unsu p ising because he u no e o he la ge , mo e complex
euka yo es’ biomass is slowe han ha o p oka yo es’ (Båå h 1998, Rousk & Båå h
2007). Acco dingly, en ichmen o euka yo ic cell componen s akes longe (Moo e e al.,
Publica ions and Manusc ip s
113
2005). Apa om a slowe u no e , he la ge size o euka yo ic cells esul s in a smalle
a io o su ace o olume. As PLFAs a e u ilized as cell memb anes on he su ace o he
o ganism and he di e ence in he a ios o
13
C in PLFA o
13
C in mic obial biomass o
a ious mic obial g oups is unknown, he e is no ull compa abili y be ween cells o di e -
en size. I is also well known ha ungi a e specialized on mo e complex subs a e han
LMWOS.
As in he euka yo es, he anae obic bac e ia also inco po a e only a maximum o
0.7% o he applied C. As he oo we ins alled p e en ed excess we ing, he soil was
well ae a ed, so he anae obic mic oo ganisms could only pe sis inside anae obic mic o-
habi a s such as mic oagg ega es. Thus, only
13
C ha pe mea ed in o hose anae obic
mic ohabi a s could be aken up by anae obic mic oo ganisms.
2.3.4.3
Disc imina ion o indi idual ca bon posi ions by mic obial u iliza ion
di e s depending on oxida ion s a e, amino acid and ime
As in soil and mic obial biomass, disc imina ion o he indi idual C posi ions o bo h
amino acids also ook place in he mic obial PLFA. As he pe cen o
13
C eco e y (Fig. 2)
be ween he mic obial g oups’ PLFA di e s g ea ly, he disc imina ion be ween he posi-
ions o alanine and glu amic acid is bes e alua ed wi h he DI (Fig. 4).
On day 3, he e was no di e ence in ela i e inco po a ion o
13
C om he me hyl
and amino-bound C o alanine o mos mic obial g oups. Nea ly no
13
C om alanine’s
ca boxyl g oup was eco e ed in he PLFAs and he inco po a ion o alanine’s
13
C in o
mic obial biomass is much lowe han ha o i s amino-bound and me hyl posi ion. Ac-
co dingly, we can conclude ha du ing he h ee days a e applying he amino acid, he
C
1
a om in alanine is spli om he molecule quickly, whe eas C
2
and C
3
emain bonded.
P esumably, he alanine molecule is aken up and hen me abolized in he main alanine
u iliza ion pa hway: deamina ion o py u a e and a e deca boxyla ion by py u a e dehy-
d ogenase, ans o ma ion o ace yl-CoA (Fig. 5a). This molecule hen ei he en e s he
ci ic acid cycle (de Kok e al., 1998) o a y acid syn hesis (Caspi e al., 2008). On day
10, he DI o he amino-bound C is sligh ly lowe han ha o alanine’s me hyl C in mos
mic obial g oups, which can be explained by he u he eac ions in mic oo ganisms: I
he molecule is used ca abolically in he ci ic acid cycle, hen he ace yl-CoA conden-
sa es wi h oxala e o ci ic acid. A e his eac ion, he o me amino-bound C o alanine
is one o ci ic acid’s ca boxyl g oups. Thus, he chance o he amino-bound posi ion o
be deg aded in o CO
2
du ing he nex s ep – he o ma ion o 2-oxoglu a a e (Camacho e
al., 1995) – is abou 1:3, whe eas he me hyl posi ion is s ill inco po a ed in he non-
eac i e chain. A e e e y ci cui o he ci ic acid cycle, C om he alanine molecule can
Publica ions and Manusc ip s
120
Supplemen a y Da a
Supplemen a y Table A1: Fa y acids in he ex e nal s anda d

Publica ions and Manusc ip s
121
Supplemen a y Table A2: Resul s o ac o analysis
Publica ions and Manusc ip s
122
2.4 S udy 4: Biogeochemical ans o ma ions o
amino acids in soil assessed by posi ion-speci ic
labeling
Michaela A. Dippold
1,2
, Yako Kuzyako
2,3
1
Depa men o Ag oecosys em Resea ch, Uni e si y o Bay eu h
2
Depa men o Ag icul u al Soil Science, Geo g-Augus -Uni e si y o Gö ingen
3
Depa men o Soil Science o Tempe a e Ecosys ems, Geo g-Augus -Uni e si y
o Gö ingen
Co esponding Au ho :
Michaela Dippold
Depa men o Ag oecosys em Resea ch
Uni e si y o Bay eu h
Uni e si ä s aße 30
95447 Bay eu h
email: [email p o ec ed]
Tel.: 0921/552187
Fax.: 0921/552246
Publica ions and Manusc ip s
123
Abs ac
BACKGROUND AND AIMS: Amino acid u no e in soil is an impo an elemen o
e es ial ca bon and ni ogen cycles. This s udy accoun s o hei d i e - he mic obial
me abolism - by acing hem ia he unique iso opic app oach o posi ion-speci ic label-
ing.
METHODS: Th ee
14
C iso opome s o alanine a i e concen a ion le els combined
wi h selec i e s e iliza ion we e used o dis inguish so p ion mechanisms, exoenzyma ic
and mic obial u iliza ion o amino acids in soil.
RESULTS: So p ion and mic obial up ake occu ed immedia ely. Unspeci ic mic o-
bial up ake ollowed a linea kine ic, whe eas ene gy-dependen up ake ollowed Micha-
elis-Men en. Less han 6% o he ini ially added alanine was so bed o soil, bu a e mi-
c obial ans o ma ion p oduc s we e bound o he soil ma ix a highe p opo ions (5-
25%). The ca boxyl g oup (C-1) was apidly oxidized by mic oo ganisms, whe eas C-2
and C-3 posi ions we e p e e en ially inco po a ed in o mic obial biomass. Dependency o
C me aboliza ion on amino acid concen a ion e lec ed indi idual alanine ans o ma ion
pa hways o s a a ion, main enance and g ow h condi ions.
CONCLUSIONS: This s udy demons a es ha posi ion-speci ic labeling de e -
mines he mechanisms and a es o C cycling om indi idual unc ional g oups. This ap-
p oach e lec ed unde lying me abolic pa hways and e ealed he o ma ion o new o -
ganic ma e . We he e o e conclude ha posi ion-speci ic labeling is a unique ool o
de ailed insigh s in o submolecula ans o ma ion pa hways and hei egula ion ac o s.
Keywo ds
: Posi ion-speci ic ace s, Amino acids s abiliza ion, So p ion, Exoenzyme and
up ake kine ics, Me abolic acing, Soil o ganic ma e o ma ion, S e iliza ion and inhibi-
ion me hods, Biochemical pa hways
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124
2.4.1 In oduc ion
S udies on ans o ma ion o o ganic subs ances in soils a e impo an o unde -
s anding o C and N cycles in e es ial ecosys ems. Plan esidues and hizodeposi s
a e he main sou ces o o ganic ma e in soils (Rasse e al., 2005). The e o e, many
s udies ha e ocused on decomposi ion, mic obial u iliza ion and s abiliza ion o C om
hese sou ces ( on Lue zow e al., 2006).
Du ing decomposi ion o li e , mac omolecula compounds a e depolyme ized by
enzymes in o low molecula weigh o ganic subs ances (LMWOS) (Cadisch and Gille ,
1996). LMWOS a e he ligh es (<250 Da) componen s o DOC (Boddy e al., 2007) om
subs ance classes such as o ganic acids, amino acids, mono- and disacca ides, amino
suga s, phenols and many mo e ( an Hees e al., 2005a). In addi ion o li e , hizode-
posi ion is a sou ce o LMWOS in soil. Mic oo ganisms de e mine he a e o LMWOS in
soil because hey ei he p oduce hem, decompose hem o CO
2
and NH
4+
(ca abolism)
o inco po a e hem in cellula compounds (anabolism). The impo ance o LMWOS is no
connec ed wi h hei pool size (Fische e al., 2007), bu wi h he huge luxes ha pass
h ough his pool. The e o e, he ans o ma ion pa hways o LMWOS ep esen a c ucial
s ep o soil C and N luxes, and a molecula -le el knowledge o hese p ocesses is
needed ( an Hees e al., 2005a).
Wi hin he LMWOS, amino acids play an impo an ole because hey a e he quan-
i a i ely mos impo an compounds coupling he C and N cycle. In opsoil, amino bound
N cons i u es 7-50% o he o al o ganic N (Ga denas e al., 2011; S e enson, 1982a).
Thus, many ecen s udies ocused on he a e o N-con aining LMWOS (Hobbie and
Hobbie, 2010; Jones e al., 2004b; Knowles e al., 2010; Kuzyako , 1996; Lipson e al.,
2001; Vinolas e al., 2001a) and in es iga ed he h ee majo pa hways o amino acid
u iliza ion in soil: 1) so p ion (Jones, 1999), 2) ex acellula ans o ma ion, and 3) in a-
cellula me aboliza ion (Vinolas e al., 2001a; Vinolas e al., 2001b) which can be sepa-
a ed by selec i e inhibi ion o bio ic p ocesses.
So p ion s ongly depends on he unc ional g oup o he amino acid (Jones and
Hodge, 1999): i can occu by ion exchange o posi i ely cha ged amino g oups, by ligand
exchange o ca boxyl g oups and by hyd ophobic in e ac ions wi h alkyl g oups. To da e,
nea ly all s udies assumed so p ion o he en i e molecule by soil so ben s. Only a ew
s udies on glycine so p ion indica ed abio ic deg ada ion o he so bed amino acid (Wang
and Huang, 2003, , 2005).
Amino acids can be ans o med ex acellula ly, mainly by exoenzymes a ached o
cell su aces (Geissele e al., 2010). Deaminina ing (Killham, 1986) and oxidizing
(Bohme e al., 1989; B aun e al., 1992) ex acellula sys ems a e desc ibed in he li e a-
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125
u e, bu nei he hei ele ance no he di e ences be ween ex a- and in acellula pa h-
ways ha e been in es iga ed (Bu ns, 1982).
In acellula amino acid me aboliza ion ollows he up ake by anspo sys ems
(An aku, 1980; Hedige , 1994; Hosie and Poole, 2001). Up ake kine ics o some amino
acids has al eady been elucida ed (Vinolas e al., 2001a; Vinolas e al., 2001b). Ba a-
clough (1997) showed ha he majo i y o N mine aliza ion o amino acids occu ed inside
he cells. Knowles e al. (2010) desc ibed o he i s ime he decoupling o N and C me-
aboliza ion in soil, disco e ing a p e e en ial e en ion o amino acid N wi h espec o C.
None heless, as hey used uni o mly labeled ace s, hey could no de e mine he a e o
he C skele on. We hypo hesize ha he a e o amino acid C and N in soil is mainly de-
e mined by he domina ing in acellula me aboliza ion pa hways. The e o e, iden i ica-
ion o mic obial me aboliza ion is a c ucial s ep o unde s anding and p edic ing C and N
luxes.
In addi ion o abio ic ac o s such as empe a u e (Dijks a e al., 2011c; Vinolas e
al., 2001b) o soil p ope ies (Gonod e al., 2006; Kemmi e al., 2008), he concen a ion
o a subs a e is a key d i e o he in acellula me aboliza ion (Dijks a e al., 2011a;
Fische and Kuzyako , 2010b; Schneckenbe ge e al., 2008). Soil amino acid concen a-
ions ange om 0.5 µM in oo - ee bulk soil o 5 mM di ec ly nex o bu s ing cells
(Fische e al., 2007; Jones and Hodge, 1999). We expec cellula up ake and me abo-
lism always domina e he amino acid emo al om soil solu ion and ha so p ion only
plays a ele an ole a low subs a e concen a ions. Fo ou s udy, we chose alanine as
a ep esen a i e amino acid o he neu al amino acids as i is one o he mos dominan
amino acids in soil solu ion (Fische e al., 2007). In addi ion, alanine was chosen be-
cause i is e y close o he basic C me abolism o he cell: by oxida i e deamina ion
alanine can be ans e ed o py u a e, which is a sui able subs a e o me abolic acing
expe imen s in plan s and soils (Dijks a e al., 2011a; Tche kez e al., 2005).
To elucida e in a- and ex acellula alanine ans o ma ion pa hways, we used he
app oach o posi ion-speci ic labeling. This ool is commonly used in biochemis y o in-
es iga e me aboliza ion pa hways, bu has a ely been applied in soil science (Dijks a e
al., 2011a; Dijks a e al., 2011b; Dijks a e al., 2011c; Fische and Kuzyako , 2010b;
Fokin e al., 1993, , 1994; Haide and Ma in, 1975; Kuzyako , 1997; Nasholm e al.,
2001). I o e comes he limi a ions o uni o m labeling because i allows di e en ia ing
be ween inco po a ion o agmen s s. inco po a ion o en i e molecules.
Coupling o posi ion-speci ic labeling wi h soil s e iliza ion enables us o sepa a e
abio ic spli ing o alanine om ex acellula and om cellula me abolism. We assume
ha ex a– and in acellula ans o ma ion di e om each o he as hey a e based on
di e en enzymes. By compa ison o he kine ics o alanine emo al om soil solu ion in

Publica ions and Manusc ip s
126
he non-inhibi ed and espi a ion-inhibi ed ea men s, he ele ance o ex a- e sus in-
acellula ans o ma ions o alanine was compa ed. We hypo hesize ha unde soil con-
di ions mic obial up ake sys ems and in acellula me aboliza ion domina e he a e o
alanine in soil. Compa ing ou esul s wi h known mic obial me aboliza ion pa hways en-
ables he iden i ica ion o me abolic changes depending on subs a e concen a ion.
2.4.2 Ma e ial and Me hods
2.4.2.1
Soil
Topsoil (0-10 cm) om he Ap ho izon o a sil loam haplic Lu isol (WRB, 2006)
was collec ed om a ield in Ba a ia wi h a c op sequencing o ba ley, whea and i icale
(49.907 N, 11.152 E, 501 m asl, mean annual empe a u e 6-7 °C, mean annual p ecipi-
a ion 874 mm). The soil had a pH
KCl
o 4.88 and pH
H2O
o 6.49, o al o ganic C and o al
N con en we e 1.77% and 0.19%, espec i ely, and po en ial CEC was 13.6 cmol
c
kg
-1
.
Soil was sie ed o 2 mm, and all oo s we e emo ed wi h weeze s. Soil was s o ed a
ield mois u e a 5 °C no longe han one week un il he expe imen s a ed.
2.4.2.2
Chemicals and adiochemicals
S ock solu ions wi h 1, 10, 100, 1000, and 10000 µM alanine and an equal ac i i y
o 10
4
DPM ml
-1
(Disin eg a ions Pe Minu e and ml) we e p epa ed om U-
14
C-labeled
alanine and he posi ion-speci ically labeled iso opome s 1-
14
C-, 2-
14
C- and 3-
14
C-labeled
alanine (Ame ican Radiolabeled Chemicals Inc, S . Louis, USA) as well as non-labeled
alanine (Sigma-Ald ich, Tau ki chen, Ge many).
S e iliza ion solu ions we e p oduced wi h 1 mM NaN
3
o inac i a e ae obic mic o-
bial espi a ion o wi h 1 mM NaN
3
and 1 mM HgCl
2
o dena u a e all p o eins and each
ull inhibi ion o bio ic p ocesses. E ec i eness o he chosen azide inhibi o was e alu-
a ed by a quali a i e 2,3,5- iphenyl e azoliumchlo ide incuba ion (TCC, Sigma-Ald ich,
Tau ki chen, Ge many). The e o e 0.63 µg o he yellow dye TCC we e added o he 1 ml
o soil suspension in his p eexpe imen .
2.4.2.3
Expe imen al se up
The e ec s o wo ac o s on alanine ans o ma ions in soil we e in es iga ed: 1)
he concen a ion o alanine, and 2) he ex a- and in acellula as well as abio ic p oc-
esses o alanine emo al om soil solu ion, sepa a ed by s e iliza ion. The e o e, h ee
Publica ions and Manusc ip s
127
s e iliza ion ea men s we e used (Fig. 1): 1) ea men s wi hou any inhibi ion, whe e
h ee g oups o p ocesses occu ed: in acellula me abolism, ex acellula ans o ma ion
and physicochemical so p ion, 2) ea men s wi h inhibi ion o ae obic espi a o y chains
by azides (Bu ns, 1982), whe e only ex acellula p ocesses a e ac i e and so p ion could
occu , and 3) ea men s wi h ull inhibi ion, whe e mic obial me abolism as well as
exoenzymes we e inhibi ed by HgCl
2
(S e enson and Ve bu g, 2006; Wol e al., 1989)
and only so p ion could emo e alanine om he soil solu ion (Fig. 1). We de ine he e as
ex acellula ans o ma ions all p ocesses (decomposi ion, deca boxyla ion, condensa-
ion, e c.) localized in he soil solu ion o pe iplasm (Glenn, 1976) which don’ depend on
in acellula ene gy me abolism (i.e. p o on g adien o ATP) and can no be inhibi ed by
NaN
3
. Bio ic ans o ma ions sums up ex a- as well as in acellula p ocesses.
Fig. 1 Scheme o he expe imen al design o one o he i e concen a ions: in pa 1
on he le side (incuba ion expe imen ) yellow-shaded pla es shows ully-
inhibi ed ea men o in es iga e so p ion whe eas g een-shaded pla es e lec
bio ic u iliza ion (uppe line wi h only ex acellula ac i i y and lowe line wi h ex-
a- and in acellula ac i i y). Yellow-shaded g aphs demons a e he calcula ion
o he so bed p opo ion o alanine by he so p ion iso he m, which is de i ed
om he ully inhibi ed ea men . G een-shaded g aphs e eal he calcula ion o
he bio ic u iliza ion by subs ac ing he so p ion om he pe cen age o alanine
emo al om supe na an . In pa 2 on he igh side (ex ac ion expe imen )
pu ple-shaded pla es e lec he ully-inhibi ed ea men and hus ex ac ion o
un ans o med alanine by he sequen ial p ocedu e. Blue-shaded pla es show
deso p ion o bio ic alanine ans o ma ion p oduc s (uppe pla e wi h only ex-
acellula ac i i y and lowe pla e wi h ex a- and in acellula ac i i y).
Publica ions and Manusc ip s
128
The expe imen consis ed o wo pa s (Fig. 1): In he i s pa – he incuba ion ex-
pe imen - he p ocesses emo ing alanine om he supe na an we e in es iga ed. The
incuba ion was pe o med in 24-deep-well pla es (6 ml olume pe well) on a o a ional
shake a 200 pm wi h 200 mg ield esh soil pe eplica ion. Be o e adding he alanine,
he soil was p e-incuba ed o 1 h wi h 0.5 ml o 1 mM s e iliza ion solu ions o dis illed
wa e , espec i ely. P e-incuba ion was pe o med unde in ensi e shaking o enable a
homogenous s e iliza ion o he en i e soil olume unde high oxygen supply. Thus, du -
ing p e-incuba ion anae obic p ocesses we e p e en ed, he s o ed ene gy could be con-
sumed and no new ene gy ese es we e p oduced.
In he ea men wi h ex acellula p ocesses, he in acellula espi a ion was inhib-
i ed wi h 0.5 ml 1 mM NaN
3
. Al hough chosen NaN
3
-concen a ions a e a abo e hose
desc ibed o espi a o y chain inhibi ion (Ki a e al., 1984) some ac i i y may emain in
he soil suspension. This was e alua ed by a iphenyl- e azolium chlo ide assay. This
dye is in acellula ly educed by a ious dehyd ogenases (K asniko e al., 1974;
Mohammadzadeh e al., 2006). An ac i e in acellula me abolism leads o he o ma ion
o insoluble ed o mazan c ys als wi hin li ing cells. In he ea men wi h ull inhibi ion,
dena u a ion o p o eins was achie ed by adding 0.5 ml o 1 mM HgCl
2
and 1 mM NaN
3
.
A e p e-incuba ion, 0.5 ml o he alanine-solu ion was added. All expe imen s we e
pe o med wi h uni o mly labeled alanine and he h ee iso opome s. The soil suspension
was shaken o 30 seconds, cen i uged a 2000 pm and an aliquo o 50 µl was emo ed
o
14
C measu emen . A e emixing, incuba ion was con inued, and u he 50 µl we e
sampled 5, 15, 30 and 60 min and 6 h, 12 h and 36 h a e addi ion o
14
C labeled
alanine. A e incuba ion, he emaining supe na an was emo ed and soil was washed
h ee imes – i s wi h dis illed wa e , hen wi h ull inhibi ion solu ion and inally wi h dis-
illed wa e .
In he second pa o he s udy - he deso p ion expe imen - we e alua ed he bind-
ing mechanisms o alanine C in soil (Fig. 1). In ea men s wi h ull inhibi ion, he ex-
ac ed C e lec s alanine C i sel , as no bio ic ans o ma ion occu ed. In ea men s wi h
bio ic ac i i y, he mic obial o ex acellula ans o ma ion p oduc s we e ex ac ed. The
washing s ep wi h HgCl
2
led o dena u a ion o memb ane p o eins and hus a loss o
memb ane in eg i y. This allowed he join ex ac ion o wa e soluble cy oplasm com-
pounds and ex acellula ans o ma ion p oduc s. Mac omolecula compounds like p o-
eins, polysaccha ides o pep idoglycan as well as hyd ophobic compounds like he
memb ane lipids could no be ex ac ed by a sal solu ion.
Fo he deso p ion expe imen , 0.5 ml o 0.5 M CaCl
2
solu ion was added o he soil
and shaken o 2 h. The solu ion was cen i uged, and supe na an was emo ed and
s o ed o
14
C analysis. Deso p ion was epea ed h ee imes, and he supe na an s we e
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129
combined o one solu ion, in which
14
C was analyzed. This deso p ion ea men wi h
CaCl
2
enabled e alua ing he amoun o alanine being weakly bound, mainly by ion ex-
change. A e ex ac ion wi h CaCl
2
he same p ocedu e was done h ee imes wi h 0.5 M
NaH
2
PO
4
solu ion o ex ac he alanine bound by ligand-exchange. To es ima e i e-
e sibly bound alanine C, he soil was eeze-d ied and combus ed a 600 °C o 10 min
unde a cons an O
2
s eam wi h a HT 1300 solid combus ion module o he mul i N/C
2100 analyze (Analy ik Jena, Jena Ge many).
14
CO
2
eleased by combus ion was
apped in 10 ml o 1 M NaOH. The i e e sibly bound pool con ains un ans o med, i e-
e sibly bound alanine C as well as mac omolecula , hyd ophobic o i e e sibly bound
mic obial ans o ma ion p oduc s.
2.4.2.4
Radiochemical analyses
14
C ac i i y o he supe na an s was de e mined using a scin illa ion coun e (Wal-
lac 1450, Mic oBe a
®
T iLux, Pe kinElme , Walham MA; USA) by adding 50 µl o he su-
pe na an di ec ly o 0.6 ml scin illa ion cock ail (EcoPlus, Ro h Company, Ge many) in
anspa en 24-well pla es. Remaining supe na an , washing solu ion and deso p ion so-
lu ion we e measu ed in glass scin illa ion ials wi h he LS 6500 scin illa ion coun e
(LS 6500, Beckman-Coul e , K e eld, Ge many) wi h a 1:2 a io o solu ion o scin illa ion
cock ail and a 1:8 a io o he CaCl
2
and NaH
2
PO
4
solu ions.
14
C ac i i y in he NaOH
solu ion was measu ed wi h a 1:2 a io o sample o scin illa ion cock ail a e 24 h o da k
s o age a e disappea ance o chemoluminescence. All measu emen s wi h he LS 6500
we e also pe o med wi h blanks o he espec i e solu ions (CaCl
2
, NaH
2
PO
4
o NaOH)
and backg ound co ec ed by sub ac ing his alue om each measu emen esul .
2.4.2.5
Calcula ion o he kine ics o alanine u iliza ion
To calcula e he bio ic u iliza ion he amoun o so bed alanine C has o be sub-
ac ed om he o al emo al om soil suspension. The e o e, he dec ease in
14
C ac i -
i y in he supe na an o he ully inhibi ed ea men (A
%
( ) in pe cen o added ac i i y)
was i ed o an exponen ial equa ion (Fig. 1) whe e B (% o added ac i i y) and c (1/h)
a e he i ed pa ame e s and D
equ
is he emaining pe cen age o ac i i y in he supe -
na an a equilib ium. The emaining ac i i y D
equ
was con e ed in o he amoun s o
so bed alanine pe g soil (S
equ
in µmol g
-1
) and he dissol ed alanine concen a ion c
equ
(µM) we e calcula ed and all i e concen a ion ea men s we e i ed by a linea ized
F eundlich so p ion iso he m wi h he so p ion a ini y cons an s k and n (Fig. 1). Based
on Fische and Kuzyako (2010b), he i ed so p ion iso he m was used o calcula e he
Publica ions and Manusc ip s
232
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Supplemen a y Da a
Supplemen a y Table A1: Sol en g adien s and low condi ions o he IC-O-IRMS
measu men
ime 20 mM NaOH 200 mM Na-
OH H
2
O 0.01 M
NaNO
3
low
(ml min
-1
)
-25 min 0% 100% 0% 0% 0.400
-10 min 8% 0% 92% 0% 0.325
11 min 40% 0% 35% 25% 0.400
15 min 45% 0% 45% 10% 0.400
18 min 25% 25% 50% 0% 0.380
35 min s andby
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2.8 S udy 8: Fo ma ion and ans o ma ion o a y
acids in soil assessed by posi ion-speci ic label-
ing o p ecu so s
Michaela Dippold
1,2
, Yako Kuzyako
1,3
1
Depa men o Ag icul u al Soil Science, Geo g-Augus -Uni e si y o Gö ingen
2
Depa men o Ag oecosys em Resea ch, Uni e si y o Bay eu h
3
Depa men o Soil Science o Tempe a e Ecosys ems, Geo g-Augus -Uni e si y o Gö -
ingen
Co esponding Au ho :
Michaela Dippold
Depa men o Ag icul u al Soil Science
Geo g-Augus Uni e si y o Goe ingen
Buesgenweg 2
37077 Goe ingen
email: [email p o ec ed]
Tel.: 0551-3933546
Fax.: 0551-3933310
Publica ions and Manusc ip s
237
Abs ac
Fa y acids a e equen ly used as plan and mic obial bioma ke s o ace he
pa hways o C s abiliza ion and soil o ganic ma e (SOM) o ma ion. Whe eas mic obial
and plan a y acid inge p in s a e well in es iga ed, hei ans o ma ions in soils emain
unclea . Howe e , knowledge o he ans o ma ion pa hways in soils is c ucial o he
in e p e a ion o a y acid inge p in s, especially because he o ma ion and decomposi-
ion p ocesses a e simul aneously ongoing. The e o e, we analyzed he o ma ion o mi-
c obial a y acids om hei p ecu so ace a e and he ans o ma ion o palmi a e in soil
by coupling posi ion-speci ic
13
C labeling wi h compound-speci ic
13
C analysis.
Posi ion-speci ically and uni o mly
13
C-labeled ace a e and palmi a e we e applied
in an ag icul u al Lu isol. Pa hways o a y acids we e aced by analyzing mic obial u ili-
za ion o C om indi idual molecule posi ions o ace a e and palmi a e and hei inco po-
a ion in o phospholipid a y acids (PLFAs).
Ace a e
13
C inco po a ion in o mic oo ganisms and ha emaining in he soil we e
cha ac e ized by basic mic obial me abolism: C-1 is p e e en ially oxidized o CO
2
in he
ci ic acid cycle, whe eas C-2 is p e e en ially inco po a ed in o mic obial compounds. I
palmi a e was used in basic C me abolism, i was spli in o C2-uni s (ace yl-CoA), and
odd and e en posi ions o palmi a e we e ans o med in a manne simila o ace a e.
Howe e , as palmi a e is he p e e ed p ecu so o PLFA o ma ion, mo e han 6% o
he added palmi a e was inco po a ed in o mic obial cell memb anes. Newly o med a y
acids we e i s , on day 3, domina ed by basic, s aigh chain a y acids. Wi h inc easing
ime, he pa e n o newly o med PLFA app oached he inge p in o he mic obial com-
muni y. The e o e, he C backbone o palmi a e was no spli , bu modi ied (e.g. desa u-
a ed, elonga ed o b anched) acco ding o he a y acid demand o he soil mic obial
communi y. I ace a e
13
C was used o PLFA o ma ion, he cons uc ion o new C back-
bones o a y acids a ely occu ed. Howe e , ace a e
13
C was inco po a ed in o mic o-
bial PLFAs by elonga ions o b anchings o al eady exis ing a y acids. The e o e, he
p e ious assump ion, ha a y acids a e gene ally newly o med om he added sub-
s a es has o be disca ded and u u e PLFA s udies ha e o conside he euse o exis -
ing plan and mic obial-de i ed a y acids.
Disc imina ion o ace a e posi ions by PLFA o ma ion was lowes in he mic obial
g oups wi h he highes compe i i eness o ace a e up ake. In con as , palmi a e up ake
and ans o ma ions we e highly speci ic o he indi idual mic obial g oups in soil. Fo
bo h subs a es, i could be concluded ha mo e di ec , less complex me abolic pa hways
a e cha ac e is ic o as -g owing mic obial g oups wi h high u no e .

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This s udy p o es he as mic obial u no e o he ee a y acid pool in soils, as
well as he high u no e and ans o ma ion o cellula PLFAs. Knowledge abou hese
mic obial ans o ma ions o a y acids in soils is c ucial o he in e p e a ions o mic o-
bial as well as plan -de i ed a y acid inge p in s. Fu he mo e, acing he o ma ion and
ans o ma ion o lipids in soils imp o es ou unde s anding o C luxes and he s abiliza-
ion o mic obial as well as plan -de i ed lipids in soils.
Keywo ds:
posi ion-speci ic ace s, me abolic acing, bioma ke app oaches, a y acid
o ma ion and ans o ma ion, phospholipids, lipid s abiliza ion, paleoen i onmen al e-
cons uc ions
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239
2.8.1 In oduc ion
Soil o ganic ma e (SOM) is he la ges ac i e ca bon (C) pool (1462-1548 Pg,
(Ba jes, 1996)) wi hin he global ca bon (C) cycle, bu he genesis and ans o ma ion
p ocesses a e poo ly unde s ood. The main inpu o C in o soils occu s ia plan li e o
hizodeposi ion (Rasse e al., 2005). Li e is composed o mac omolecules such as cellu-
lose, hemicellulose, lignin o p o eins (C aw o d e al., 1977; So ensen, 1975); in addi ion,
hizodeposi s con ain low molecula weigh o ganic subs ances (LMWOS) (Fa a e al.,
2003). Along wi h wa e soluble low molecula weigh o ganic compounds and hei poly-
me s, lipids a e impo an cons i uen s o plan biomass. They comp ise a ound 3-10% o
abo eg ound and 0.5-5% o belowg ound plan biomass and a e hus an essen ial com-
pound o plan C inpu in o soils (Bliss, 1962; Ohl ogge and B owse, 1995; Wiesenbe g e
al., 2004). In addi ion, mic obial biomass con ains a ound 10% o lipids, mainly in hei
cell memb anes and cell walls (Lengele e al., 1999; Zelles e al., 1995) and signi ican ly
con ibu e o he lipidic SOM pool.
Lipids comp ise a highe pe cen age o SOM (Almend os e al., 1991; Rumpel e
al., 2004) han hei sou ce ma e ial i.e. plan and mic obial biomass. This accumula ion
o lipids al eady indica es hei selec i e p ese a ion in soils (Lich ouse e al., 1998a).
Mo eo e , lipids a e assumed o play an e en mo e impo an ole in SOM o ma ion and
s abiliza ion: lipids a e s abilized by hyd ophobic in e ac ions wi h SOM and wi h each
o he , leading o a dec eased we abili y and subsequen hampe ing mic obial decompo-
si ion (Lich ouse e al., 1998a; on Lue zow e al., 2006). In addi ion, unc ional g oups o
lipids can co alen ly bind u he molecules (Alla d, 2006; Be hie e al., 2000) o encap-
sula e smalle molecules, leading o hei p ese a ion (Lich ouse e al., 1998b; Piccolo,
2002; Su on and Sposi o, 2005).
The long- e m p ese a ion o some lipid classes quali ies hem as impo an bio-
ma ke s (O o e al., 2005; Whi e e al., 1997). N-alkanes a e commonly assumed o be
plan -de i ed (Kuhn e al., 2010; Lich ouse, 1998) and a e used as plan bioma ke s o
di e en ia e ege a ion ypes (Bush and McIne ney, accep ed 2013; Schwa k e al., 2002;
Zech e al., 2012); long-chain a y acids a e used in a simila way (Wiesenbe g and
Schwa k, 2006). Mo e complex cu in-sube in-de i ed hyd oxyla ed o poly-ca boxylic
acids enable abo eg ound li e inpu o be di e en ia ed om belowg ound li e inpu
(Mendez-Millan e al., 2011; Spiel ogel e al., 2010). S e ols and o he isop enoid lipids,
like e penoids, a e indica i e o animal o plan -de i ed SOM (O o and Wilde, 2001).
Applica ion o all hese bioma ke s aci ly assumes ha hey emain unmodi ied in
soils o e long pe iods. Howe e , mic oo ganisms a e able o use lipids as subs a es
and decompose hem o me aboli es as well as build up hei own lipids, he e o e signi i-
Publica ions and Manusc ip s
240
can ly con ibu ing o he lipid pool o SOM and he modi ica ion o ini ial lipidic com-
pounds (Lich ouse e al., 1995; O o e al., 2005). This indica es al eady ha he concep
o gene ally un ans o med lipid bioma ke s is likely o be oo simpli ied. Recen s udies
indica e ha plan -de i ed bioma ke s can be modi ied and o e p in ed by hizomic obial
ac i i y (Gocke e al., in p ess). Fo some bioma ke classes like s e ols mic obial modi i-
ca ions o plan o animal-de i ed lipids a e speci ically used o ace he mic obial com-
muni y impac (A ima e al., 1969; Bull e al., 2002; Bull e al., 1999). Fo o he s, like al-
kanes, app oaches used o co ec o mic obial o e p in o plan -de i ed signals ha e
been de eloped (Buggle e al., 2010; Zech e al., 2013). Howe e , cu en knowledge on
he mic obial ans o ma ion o lipids in soils is a e and new expe imen al s udies a e
needed.
14
C age o mic obial lipid bioma ke s e ealed ha hey a e no indica i e o he
age o his subs ance class, as mic oo ganisms ob iously use old lipidic subs a es o
build up hei new lipids, e.g. phospholipid a y acids (PLFA) (Re hemeye e al., 2004).
Howe e , i is no ye clea whe he mic oo ganisms p e e he new syn hesis o hei lip-
ids om low molecula weigh p ecu so s like ace a e o whe he hey p e e o use a ail-
able lipidic compounds, e.g. a y acids, and simply modi y hem. Acco ding o he bio-
chemical p inciple o he mos economic pa hways, cells end o use p e o med building
blocks o biomass syn hesis (Lengele e al., 1999). Consequen ly, we hypo hesize ha
lipids eleased by he decomposi ion o plan o mic obial biomass should be he p e-
e ed subs a es o u he lipid syn hesis by mic oo ganisms. Howe e , i is known ha
mic obial pola lipids, like PLFA, a e deg aded o a y acids a e cell dea h (Lich ouse e
al., 1995) and, consequen ly, e-con ibu e o he ee lipid pool in soils. The knowledge
gap conce ning he soil lipid cycle has caused an in ensi e discussion conce ning he
con ibu ion o plan e sus mic obial lipids (Lich ouse, 1998; Lich ouse e al., 1995; O o
e al., 2005). The e o e, mic obial modi ica ion o lipid bioma ke s in soil, as well he
ans o ma ion p ocesses, a e c ucial o he applica ion o bioma ke inge p in s as well
as iso ope da a om lipid bioma ke s.
The e o e, we aced he mic obial o ma ion o memb ane lipids, PLFA, om hei
low molecula weigh o ganic p ecu so – he ace a e (Caspi e al., 2008; Kesele e al.,
2009; Rock e al., 1981). In addi ion, we aced he u iliza ion o he mos abundan a y
acid – palmi ic acid – as a mic obial subs a e o PLFA. Palmi ic acid is a key compound
o plan and mic obial a y acid me abolism. In es iga ing i s mic obial u iliza ion and
ans o ma ion pa hways e eals a gene al iew o he mic obial modi ica ion o soil lipids.
To elucida e he me abolic pa hways o mic obial a y acids, we used he app oach
o posi ion-speci ic labeling. This ool was o iginally de i ed om biochemis y o in es i-
ga e me abolism pa hways and has a ely been applied in soil science (Fokin e al., 1993,
Publica ions and Manusc ip s
241
, 1994; Haide and Ma in, 1975; Kuzyako , 1997; Nasholm e al., 2001). Howe e , wi hin
he las yea s, inc easing in e es in he use o posi ion-speci ic labeling o assess me a-
bolic pa hways in soil has a isen (Apos el e al., 2013; Dijks a e al., 2011a; Dijks a e
al., 2011b; Dippold and Kuzyako , 2013; Fische and Kuzyako , 2010b). This is because
his app oach is he only one which enables he a e o indi idual C posi ions o be aced
h ough a ious pools o me aboli es and consequen ly allows he econs uc ion o indi-
idual ans o ma ion s eps.
Knowledge abou a y acid syn hesis by mic oo ganisms is mainly de i ed om ex-
pe imen s wi h pu e cul u es (Lenna z, 1970; Rock e al., 1981; Zelles e al., 1995). They
can be newly syn hesized om p ecu so s like ace a e o a ailable lipid p ecu so s can
be modi ied by hem (Lenna z, 1970; Re hemeye e al., 2004; Rhead e al., 1971). I
palmi a e is gi en as a subs a e, he e a e h ee possible mechanisms by which palmi-
a e C can be used o PLFA syn hesis: 1) he esyn hesis pa hway i.e. he comple e deg-
ada ion o he molecule o ace yl-CoA uni s and he ollowing econs uc ion o new a y
acids om C2-mojie ies (Rhead e al., 1971); 2) Pa ial s ep-by-s ep deg ada ion o he
C2-uni s wi hou o al b eakdown o palmi a e: subsequen ly, only pa s o he molecule
a e inco po a ed in o newly o med a y acids (Rhead e al., 1971); and 3) he un ans-
o med u iliza ion o palmi a e as i is he mos abundan a y acid in mic oo ganisms
(Rhead e al., 1971; Zelles e al., 1995). Posi ion-speci ic
13
C labeling enables hese h ee
pa hways o be dis inguished and o e alua e he ans o ma ion o he s aigh chain,
unsa u a ed palmi a e. This app oach will deepen he unde s anding o he ans o ma ion
o a y acids and o he lipidic bioma ke s and imp o e he in e p e a ion o a y acid in-
ge p in s in soils.
2.8.2 Ma e ial and Me hods
2.8.2.1
Expe imen al Si e
The ield expe imen is loca ed in Ba a ia, close o Hohenpölz (49.907 N, 11.152 E)
wi h 501 m.a.s.l, mean annual empe a u e 6.7 °C and mean annual p ecipi a ion o
874 mm. The ag icul u ally used ield si e is managed by a o a ion o co n, ba ley, whea
and i icale. Soil ype is a loamy Lu isol which has a pH
KCl
o 4.88, a pH
H2O
o 6.49, a
TOC con en o 1.77%, a TN con en o 0.19% and a CEC o 13 cmol
C
kg
-1
. Be o e he
expe imen s a ed in Augus 2010 i icale, he las c op, was ha es ed, and he ield si e
was g ubbed o soil homogeniza ion.
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Fig. 2 Classes o phospholipid-de i ed a y acids ( op le ) ex ac ed om soil and
eco e y o posi ion-speci ically
13
C-labeled ace a e ( op igh ) and palmi a e
(bo om) in he di e en a y acid classes 3 and 10 days a e
13
C applica ion.
Expe imen al poin s (means ± SEM, N=4) a e p esen ed.
Figu e 2 shows ha ace a e was an app op ia e p ecu so o he new o ma ion o
a y acids: e.g. a day 3, he pa e n o newly o med a y acids om ace a e C-2 was
al eady qui e simila o he PLFA p o ile in soils (Figu e 2). Only b anched a y acids
smalle han 16 C a oms we e no o med om ace a e, no e en om ace a e C-2. In
gene al, ace a e C-1 inco po a ion in o each a y acid was lowe han C-2, e lec ing ha
no only in ac ace a e was used as a p ecu so in a y acid syn hesis. Especially in
b anched a y acids, a p e e en ial inco po a ion o C-2 was obse ed (Figu e 2). The C-
1 om ace a e was no inco po a ed in any o he odd-numbe ed a y acids (Figu e 2).
Palmi a e
13
C inco po a ion in o phospholipid-bound palmi a e is highe han ace a e
13
C inco po a ion (Figu e 2). C-1 o palmi a e is inco po a ed only in negligible amoun s
in o a y acids which we e sho e han 16 ca bons (Figu e 2 and Figu e 3). In addi ion,
he e minal C-1 posi ion o palmi a e was no inco po a ed in o odd-numbe ed PLFAs.
This indica es he p e e ence o use palmi a e as a di ec p ecu so o mic obial phos-
pholipid syn hesis.

Publica ions and Manusc ip s
249
Fig. 3 Finge p in o phospholipids-de i ed a y acids in soil ( op le ) and ela i e e-
co e y o indi idual posi ions om palmi a e
13
C in a y acid classes. Expe i-
men al poin s (means ± SEM, N=4) a e p esen ed.
Figu e 3 p esen s he ans o ma ions o inco po a ed palmi a e by mic obial PLFA
o ma ion. The po ion o desa u a ed a y acids was al eady high a day 3 and did no
inc ease signi ican ly om day 3 o day 10 (Figu e 3). This shows a as desa u a ion o
he palmi a e p ecu so . Howe e , inco po a ion o palmi a e
13
C in o elonga ed and e en
mo e in o b anched a y acids signi ican ly inc eased om day 3 o day 10 o each o he
C posi ions, e lec ing slowe kine ics o hese p ocesses.
In gene al, a modi ica ion o palmi a e acco ding o he demand o he mic obial
communi y could be obse ed in his s udy. A e 10 days he inge p in o newly o med
a y acids al eady closely app oached he PLFA dis ibu ion o he p esen mic obial
communi y. Howe e , indi idual ans o ma ion s eps occu ed wi h di e en kine ics.
2.8.3.3
Inco po a ion o ace a e and palmi a e
13
C in o PLFAs o indi idual
mic obial g oups
P e e ence o ace a e and palmi a e s ongly di e ed o indi idual mic obial
g oups. The PCA, based on he amoun s o a y acids, e ealed wo g oups o g am
nega i es: whe eas g am-nega i e 1 (18:1w9c, 18:1w7c, 14:1w5c) showed he highes
inco po a ion o ace a e, g am-nega i es 2 (16:1w7c, cy19:0) e lec ed he highes up ake
o palmi a e (Figu e 4). In gene al, he low molecula weigh ace a e was a be e sub-
s a e o p oka yo ic g oups han o euka yo ic ungi o p o ozoa. A simila pa e n was
no obse ed o palmi a e, which was p e e en ially used by p oka yo ic g am-nega i es
and euka yo ic ungi and p o ozoa (Figu e 4). The amoun o inco po a ed
13
C dec eased
om day 3 o day 10 o each o he mic obial g oups and bo h ca boxylic acids (Figu e 4,
he only excep ion is inco po a ion o palmi a e in o ac inomyce es).
Publica ions and Manusc ip s
250
Fig. 4 Reco e y o applied
13
C om posi ions o ace a e ( op) and palmi a e (bo om) in
mic obial g oups a e 3 and 10 days. Expe imen al poin s (means ± SEM, N=4)
a e p esen ed. Signi ican di e ences o inco po a ion o indi idual posi ions and
inco po a ion be ween he days, calcula ed by nes ed ANOVA, a e p esen ed in
Supplemen a y, Table A4
Speci ics in he ace a e and palmi a e ans o ma ions a e mo e isible i he di e -
gence index (DI) is conside ed a he han he absolu e
13
C inco po a ion (Figu e 5). Fo
ace a e, each o he mic obial g oups showed he p e e en ial inco po a ion o C-2. How-
e e , he disc imina ion be ween C-1 and C-2 was lowes o g am-nega i e g oups (who
had he highes absolu e inco po a ion) and highes o euka yo ic g oups (who had he
lowes ace a e
13
C inco po a ion) (Figu e 5). The DI o palmi a e did no show simila
ends: each mic obial g oup had indi idual p e e ences o inco po a ion o palmi a e
Publica ions and Manusc ip s
251
posi ions in o hei PLFAs. Simila o ace a e, he disc imina ion be ween posi ions was
lowes o he wo g am nega i e g oups. Fo many mic obial g oups, he posi ion-speci ic
p e e ences and disc imina ion be ween posi ions s ongly changed om day 3 o 10.
This e lec s an in ensi e u no e o palmi a e
13
C, e en i inco po a ed in o PLFA.
Fig. 5 Di e gence index (DI) e lec ing disc imina ion be ween C posi ions by inco po-
a ion in o indi idual mic obial g oups 3 (le ) and 10 ( igh ) days a e applica-
ion o
13
C-labeled ace a e ( op) and palmi a e (bo om). Expe imen al poin s
(means ± SEM, N=4) a e p esen ed. Signi ican e ec s o C posi ion and day on
DI, calcula ed by nes ed ANOVA, a e p esen ed in Supplemen a y Table A5.
Le e s indica e signi ican di e ences (p < 0.05 de i ed om HSD pos -hoc
es ) in he ela i e inco po a ion o he C posi ions in o one g oup.
Publica ions and Manusc ip s
252
2.8.4 Discussion
2.8.4.1
U iliza ion and u no e o ace a e and palmi a e by soil mic obial
communi y
The sho -chain low molecula weigh o ganic acids a e a well-used mic obial sub-
s a e (Jones e al., 2003). Ou s udy shows ha long chain ca boxylic acids like palmi a e
a e also good subs a e in soils and a e used in simila p opo ions by he mic obial
communi y (Figu e 1). In speci ic pa hways, such long chain ca boxylic acids can unc ion
as di ec p ecu so s o lipid o ma ion, e.g. ha o PLFA. Then, hei inco po a ion in o
mic oo ganisms can e en exceed hose o low molecula weigh subs ances (Figu e 1).
The p e e en ial oxida ion o C-1 o ace a e is in acco dance wi h p e ious s udies
and can clea ly be linked o mic obial me abolism, i.e. he oxida ion o ace a e in he ci ic
acid cycle (Dippold and Kuzyako , 2013; Fische and Kuzyako , 2010b) (Figu e 6). A
simila p e e en ial oxida ion o C-1 was obse ed o palmi a e, especially a e 10 days.
This e lec s ha i palmi a e is used in basic C me abolism, e.g. as an ene gy sou ce, i is
successi ely oxidized by a y acid
β
-oxida ion o ace yl-CoA (2 C a oms) uni s (Caspi e
al., 2008; Kesele e al., 2009). Consequen ly, e minal C-1 and C-2 om palmi a e o m
an ace a e uni , and a e ans o med simila ly o ace a e in basic C me abolism.
Fig. 6 Me abolic pa hways o a y acid o ma ion om ace a e and a y acid ans o -
ma ions o palmi a e in soil.
A day 10, a highe po ion o he
13
C emaining in soil is ound in mic obial biomass
o ace a e han o palmia e (p<0.05). A highe po ion o he
13
C in mic obial biomass o
ace a e han o palmia e (p<0.05) clea ly shows he lowe mic obial a ailabili y o palmi-
a e. Consequen ly, a highe ela i e p opo ion o he added
13
C emained ex acellula ,
e.g. as SOM-associa ed palmi a e. Ne e heless, a high disc imina ion be ween C-1 and
C-2 was obse ed, which was e en highe o palmi a e han o ace a e a day 10. This
indica es ha in addi ion o he mic obial ans o med palmi a e also palmi a e s abilized
in soil ge s ans o med by e minal oxida ion. This e minal oxida ion o ca boxylic acids
Publica ions and Manusc ip s
253
o odd and e en alkanes has been p e iously desc ibed o plan s and mic oo ganisms
(Dennis and Kola ukudy, 1992; Ladygina e al., 2006; Pa k, 2005), and speci ic as well
as unspeci ic deca boxylases con ibu e o he deca boxyla ion o ca boxylic acids in soils
(Ho ich e e al., 1998). Ex acellula ans o ma ions a e known o be less ele an o
well a ailable, low molecula weigh o ganic subs ances (Dippold and Kuzyako , 2013).
Howe e , hei quan i a i e ele ance o hyd ophobic subs ances, such as palmi a e,
which could be s abilized by hyd ophobic in e ac ions in soils and a e consequen ly less
a ailable o mic obial up ake, s ill emains open. To inally iden i y ex acellula , e minal
oxida ion, selec i e inhibi ion o mic obial, in acellula p ocesses coupled wi h posi ion-
speci ic lipid
13
C labeling has o be pe o med (Dippold and Kuzyako , 2013).
2.8.4.2
Pa hways o a y acid o ma ion om ace a e in soil
Whe eas in mic oo ganisms he C-2 om ace a e is p e e en ially inco po a ed
compa ed o C-1, his clea pa e n is less exp essed i ace a e C is used o PLFA syn-
hesis (Figu e 1). This shows ha many o he mic obial compounds in cy osol ( eleased
by he chlo o o m- umiga ion-ex ac ion) a e small wa e soluble p oduc s like ca boxylic
acids o nucleo ides de i ed om ci ic acid cycle me aboli es. Du ing he ci ic acid cycle
he C-1 o ace a e ge s oxidized in an ea ly s ep, whe eas se e al cycles a e needed un il
ace a e C-2 ge s oxidized (Figu e 6). Howe e , ace a e is a di ec p ecu so o a y acid
syn hesis, which is buil up om he C2-uni ace yl CoA (Caspi e al., 2008; Kesele e al.,
2009; Lengele e al., 1999). The e o e, he di ec o ma ion o a y acids om ace a e
would lead o an iden ical inco po a ion o bo h posi ions (Figu e 5). This iden ical inco -
po a ion was no obse ed in his s udy. Ins ead, he e a e clea speci ics o C-1 and C-2
inco po a ion in o indi idual PLFAs (Figu e 2b): The lowe inco po a ion o C-1 in o basic,
s aigh chain a y acids like palmi a e can be explained by he u iliza ion o al eady pa -
ially oxidized agmen s o ace yl CoA o a y acid syn hesis. This is simila o he me-
abolism o glucose (Dippold e al., submi ed). Al hough glucose is a di ec p ecu so o
amino suga syn hesis, glucose molecules we e ans e ed in o basic glucose C me abo-
lism and only agmen s o he pa ially oxidized and spli molecule we e subsequen ly
used o amino suga syn hesis. Simila bidi ec ional pa hways we e obse ed o ca bo-
hyd a e syn hesis: o ma ion o mic obial suga s om glycine occu ed pa allel o di ec
o ma ion om glucose and in pa allel o glucose oxida ion (De ien e al., 2007). Such
bidi ec ional pa hways can also explain he C posi ions used o a y acid o ma ion in
his s udy: ace a e was pa ially oxidized by he ci ic acid cycle and agmen s, only con-
aining C-2, we e ans e ed back om ci ic acid cycle me aboli es owa ds ace yl-CoA
o new a y acid syn hesis.

Publica ions and Manusc ip s
254
An al e na i e explana ion would be ha he majo i y o a y acids a e no newly
o med om ace yl-CoA (equal inco po a ion o C-1 and C-2). Ins ead, ace a e
13
C was
only used o pe o m ans o ma ion and modi ica ions a al eady exis ing a y acids, e.g.
elonga ions o pa ially deg aded a y acids o he in oduc ion o b anching poin s in o
molecules. Figu e 2b shows ha no ace a e C-1 is inco po a ed in any odd a y acid: his
is a he unlikely, i a y acids wi h 15, 17 o 19 C a e newly o med om ace yl-CoA (us-
ing 7, 8 o 9 uni s o ace yl-CoA). Howe e , i only a e minal ace yl-CoA is added and
a e wa ds he e minal ca boxylic g oup is spli o each an odd a y acid, his would
cause a low inco po a ion o C-2 and he absence o C-1 inco po a ion in o odd a y ac-
ids (Figu e 2b). Fo non-g owing mic obial communi ies unde main enance condi ions,
like in his s udy (see Table 1), in e nal ecycling o a y acids is likely o occu : he u ili-
za ion o di ec p ecu so s con ibu es o sa e ene gy and C (Lengele e al., 1999).
Howe e , his can only inally be p o en, i no only posi ion-speci ic labeling bu also po-
si ion-speci ic de ec ion o he iso opic label in PLFAs is pe o med.
2.8.4.3
Pa hways o a y acid ans o ma ions in soils
The highe absolu e inco po a ion o palmi a e C compa ed o ace a e C o he
o ma ion o PLFA showed ha he mo e complex and di ec p ecu so palmi a e is p e-
e ed o syn hesis and ace a e is p e e ed o ca abolism. This sugges s al eady ha
palmi a e was no ully deg aded o ace yl-CoA and new a y acids we e buil up om
ace yl-CoA acco ding o he esyn hesis pa hway (Rhead e al., 1971). Ins ead i is likely
ha modi ica ion o in ac palmi a e occu ed. Figu e 4 shows ha he ini ially added
13
C
palmi a e is successi ely ans o med o mo e di e se spec a o a y acids. Compa ing
hose ans o med a y acids wi h he PLFA inge p in o he soil (Figu e 3) shows ha
o e a pe iod o 10 days he newly ans o med a y acids a e app oaching he composi-
ion and consequen ly he demand o he mic obial communi y. Howe e , di e en kine -
ics o ans o ma ions a e clea ly shown by igu e 3: Simple desa u a ion o palmi a e oc-
cu ed apidly du ing he i s 3 days a e labeling. The ea e , he p opo ion o desa u-
a ed a y acids only ma ginally inc eased. Mo e complex, biochemical p ocesses, like
elonga ions o e en mo e b anchings, occu ed mo e slowly and he e o e he p opo ion
o hese a y acids s ongly inc eased a e day 3 .
Howe e , he use o in ac palmi a e and he ollowing modi ica ions a e in acco -
dance wi h high ecycling o exis ing a y acids in soil mic oo ganisms obse ed by ace-
a e
13
C (Figu e 2). This is con i med by he a e o indi idual palmi a e posi ions: 1) he e
is almos no C-1 and C-2 inco po a ed in e en numbe ed a y acids smalle han palmi-
a e, e.g. C14 a y acid ( e adecanoic acid). This sugges s ha he e minal ace a e (C-1
Publica ions and Manusc ip s
255
and C-2) o palmi a e is jus spli o o o m he C14 a y acid, whe eas he basic C
skele on con aining C-16 emained in ac . 2) The e is no palmi a e C-1 in odd numbe ed
a y acids, sugges ing ha he e minal C-1 is only oxidized du ing he o ma ion o odd
numbe ed a y acids om e en numbe ed palmi a e. 3) C-1, C-2 and C-16 a e inco po-
a ed in simila amoun s in desa u a ed a y acids (Figu e 2c). This sugges s ha he
unsa u a ed, s aigh chain palmi ic acid is jus desa u a ed – o elonga ed and desa u-
a ed – o he o ma ion o desa u a ed C16 and C18 a y acids.
Posi ion-speci ic
13
C labeling canno dis inguish whe he hese modi ica ions occu
as ee a y acids o bound o he PLFA. Whe eas elonga ions and sho enings need o
occu wi h a ee, non-es e i ied e minal ca boxylic g oup (Caspi e al., 2008), modi ica-
ions like 10-me hyl b anching, cycliza ion o desa u a ion a e known o be possible i
a y acids a e bound o a PLFA (Aguila e al., 1998; Lenna z, 1970). To dis inguish
ans o ma ions o ee a y acids om hose occu ing bound in PLFAs wi hin he mem-
b ane, he measu emen o iso opic label in in ac phospholipids and ee a y acids has
o be pe o med wi h much sho e ime in e als han hose chosen o his s udy. How-
e e , i espec i e o he de ailed biochemical mechanism, his s udy p o ed: 1) an in en-
si e modi ica ion and use o in ac a y acids aken up om soil, and 2) an in ensi e e-
cycling o he mic obial a y acid pool (which can occu in acellula o in e cellula a e
cell dea h). The e o e, he p e ious assump ion ha a y acids a e gene ally newly
o med om he added subs a es ha e o be disca ded and u u e PLFA s udies, be-
cause hey ha e o conside he euse o exis ing plan and mic obial-de i ed a y acids
(see sec ion 4.5).
2.8.4.4
Pa hways o speci ic mic obial g oups in soils
Fo he example o ace a e, Figu e 3 e lec s he classical use o LMWOS by indi-
idual mic obial g oups in soils: g am-nega i es a e known o be he domina ing g oup in
he hizosphe e (Sode be g e al., 2004; Tian e al., 2013) and a e mos compe i i e o
LMWOS (Apos el e al, 2013, Gunina e al, submi ed). In con as , g am-posi i es p e e
old SOM (K ame and Gleixne , 2006) and a e less compe i i e o LMWOS. In gene al,
he mo e complex o ganisms a e s uc u ed, he lowe hei u no e and hei compe i-
i eness o as up ake o LMWOS is: Bac e ia ha e a sho e gene a ion ime (bac e ia
20 min e sus ungi 4-8 h o comple e a li e cycle unde op imal condi ions) and conse-
quen ly highe cellula u no e (bac e ia 2-3 imes and ungi 0.75 imes biomass u no e
pe yea unde soil condi ions) (Moo e e al., 2005; Rousk and Baa h, 2007; Wa ing e al.,
2013). The u no e is e en slowe o highe le els o he nu i ional ne , e.g. he p o o-
zoa. Consequen ly, he esul s o ace a e u iliza ion in his s udy con i m ha as g ow h
Publica ions and Manusc ip s
256
is based on he u iliza ion o eadily a ailable subs a es and is closely associa ed wi h a
as u no e o he espec i e mic obial g oups in he soil. Mic obial g oups wi h a as e
u no e a e commonly mo e compe i i e o LMWOS, like ace a e, e en i he mic obial
communi y in gene al is unde main enance condi ions (Gunina e al., submi ed).
Howe e , such a gene al ule is no alid o mo e complex, no ubiqui ous sub-
s a es like palmi a e: Figu e 3 shows ha , e en wi hin he g am-nega i es, he e is a
clea p e e ence o palmi a e u iliza ion by he g am-nega i es 2. Sepa a ion o he wo
g oups o g am-nega i es was based on di e en g oupings o he espec i e g am-
nega i e a y acids by explo a i e s a is ical ools (he e: loadings o a y acid con en s on
di e en ac o s in a p inciple componen analysis). This ool, commonly used o cha ac-
e ize a y acid inge p in s, suppo ed he e he sepa a ion and iden i ica ion o wo eco-
physiologically di e en g oups o g am-nega i es: g am-nega i e 1 wi h a p e e ence o
LMWOS and g am-nega i e 2 wi h a p e e ence o mo e complex, hyd ophobic ca box-
ylic acids (Figu e 4).
No only he absolu e up ake bu also he me abolism was speci ic o he in es i-
ga ed mic obial g oups. The Di e gence Index e ealed he p e e ence o ace a e C-2
inco po a ion o each o he mic obial g oups. Howe e , disc imina ion be ween C-1 and
C-2 inc eased signi ican ly o hose g oups wi h high LMWOS, i.e. ace a e, up ake. (Fig-
u e 4 and Figu e 5). This can ha e wo possible easons: 1) Fas g owing mic obial
g oups wi h apid u no e a e cha ac e ized by a mo e di ec me abolismn using p ecu -
so s wi hou u he ans o ma ion, and 2) The as g owing g am-nega i es a e mainly
cha ac e ized by s aigh chain, monounsa u a ed C16 and C18 a y acids (Zelles, 1999),
which a e o med by simple desa u a ion wi hou complex me abolic p ocesses like me-
hyla ions o b anchings (Lenna z, 1970) leading o disc imina ions be ween C-1 and C-2.
In bo h cases, i can be concluded – a leas o he PLFA o ma ion pa hway - ha mo e
di ec , less complex me abolic pa hways a e cha ac e is ic o as g owing mic obial
g oups wi h high u no e . This is also con i med o palmi a e inco po a ion in o PLFA,
whe e he g am-nega i es showed a compa a i ely low disc imina ion be ween he palmi-
a e posi ions (Figu e 5). Howe e , o p o e hese gene al o mic obial g oup-speci ic
ans o ma ion s eps, a combina ion o posi ion-speci ic labeling wi h posi ion-speci ic
analysis o he mic obial ans o ma ion p oduc s is needed. Ne e heless, he s ong
di e ence in DI om day 3 o day 10 o palmi a e con i ms ha a high, in e nal u no e
e.g. by ecycling and ans o ma ion o he a y acids, ook place a e
13
C inco po a ion.
Publica ions and Manusc ip s
257
2.8.4.5
Consequences o he applica ion o a y acids as bioma ke s
The obse ed ans o ma ion o ee a y acids in soil by mic oo ganisms causes
consequences o he applica ion o a y acids as mic obial and plan bioma ke s. Al-
kanes can mo e easily be dis inguished be ween plan - and mic obial-de i ed n-alkanes.
This enables he alkane inge p in o be co ec ed o mic obial con ibu ion (Buggle e
al., 2010; Zech e al., 2013). In con as , he di e en ia ion be ween mic obial and plan -
de i ed a y acids is no as sha p: Vege a ion ype as well as mic obial communi y a ec
he a y acid sou ces in soils (O o e al., 2005) and, in many cases, a econs uc ion o
he o iginal sou ce is no possible (Gocke e al., 2014). Fu he mo e, i is no clea ,
whe he mic obial enzyma ic sys ems modi ying n-alkanoic acids like palmi a e a e highly
speci ic enzymes, which wo k only in acellula ly o whe he unspeci ic modi ica ion o
plan -de i ed ee a y acids can occu . This would s ongly limi he applica ion o a y
acid inge p in s (Zhou e al., 2005) as well as hei iso ope signa u es (Li e al., 2011) o
paleo-en i onmen al econs uc ions. The e o e, u he in es iga ions, e.g. posi ion-
speci ic labeling o long-chain plan -de i ed a y acids and in es iga ion o hei mic obial
ans o ma ions, is needed.
The ans o ma ion and in e nal ecycling o a y acids wi hin mic obial cells has
impo an consequences o i s applica ion as mic obial bioma ke s. Changes wi hin he
a y acid inge p in in soils a e commonly assumed o be ela ed o changes in he mi-
c obial communi y s uc u e (Zelles, 1999). Howe e , ace a e as well as palmi a e label-
ing showed in his s udy ha a y acids a e ans o med and modi ied e y as in soils.
Pu e cul u e s udies con i m ha hese modi ica ions o a y acids occu wi hin li ing
cells, i en i onmen al condi ions su ounding a li ing o ganism a e changing, e.g. by
empe a u e changes (Aguila e al., 1998). These modi ica ions o exis ing a y acids
can e en occu in in ac PLFA wi hin he memb anes (Aguila e al., 1998; Lenna z,
1970). The e o e, u he knowledge abou he impac o in e nal a y acid u no e o
he in e p e a ion o he PLFA inge p in is needed (F os ega d e al., 2011). Howe e ,
he high in e nal u no e o a y acids wi hin li ing mic obial cells explains he disc ep-
ancy be ween he u no e o PLFA and o ha o mic obial biomass. PLFAs a e as-
sumed o ha e a hal -li e be ween one day and one week (Kindle e al., 2009; Rannekle
and Baa h, 2003; Re hemeye e al., 2004). In con as , he u no e o he bac e ial mi-
c obial communi y is assumed o occu 2-3 imes pe yea (Moo e e al., 2005; Rousk and
Baa h, 2007; Wa ing e al., 2013). An in ensi e in acellula u no e o PLFA explains he
much as e u no e o PLFA. This is simila o obse a ions o he u no e o mic obial
cell walls (Dippold e . al, submi ed): o
E. coli
i was e en shown ha hey ecycle 60%
o hei pep idoglycan du ing cellula li e (Pa k and Ueha a, 2008; Ueha a and Pa k,
2008). Malik e al. (2013) showed ha he u no e o mic obial biomass compounds de-
Publica ions and Manusc ip s
264
Zelles, L., 1999. Fa y acid pa e ns o phospholipids and lipopolysaccha ides in he cha -
ac e isa ion o mic obial communi ies in soil: a e iew. Biology and Fe ili y o Soils
29, 111 - 129.
Zelles, L., Bai, Q.Y., Rackwi z, R., Chadwick, D., Beese, F., 1995. De e mina ion o
phospholipid-de i ed and lipopolysaccha ide-de i ed a y acids as an es ima e o mi-
c obial biomass and communi y s uc u es in soils. Biology and Fe ili y o Soils 19,
115-123.
Zhou, W.J., Xie, S.C., Meye s, P.A., Zheng, Y.H., 2005. Recons uc ion o la e glacial and
Holocene clima e e olu ion in sou he n China om geolipids and pollen in he Ding-
nan pea sequence. O ganic Geochemis y 36, 1272-1284.

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265
Supplemen a y Da a
Supplemen a y Table A1: Fa y acids in he ex e nal s anda d
Supplemen a y Table A2: Fa y acids in he ex e nal s anda d
Publica ions and Manusc ip s
266
Supplemen a y Table A3: Resul o ac o analysis
Supplemen a y Table A4: Nes ed ANOVA o ace a e and palmi a e posi ions nes ed in
he a iable day, block as andom a iable and day. Deg ees o eedom (d ), F- alues
and signi icance le el (p) a e shown o he ace a e and palmi a e. I equi emen s o
pa ame ic es s (no mal dis ibu ion + homogenei y o a iances was no gi en, a
K uskal-Wallis ANOVA o he indi idual ea men s was calcula ed (in his case H-Value
is gi en ins ead o F alue)
Publica ions and Manusc ip s
267
Supplemen a y Table A5: Nes ed ANOVA o ace a e and palmi a e DI, wi h he inde-
penden a iables posi ion (being nes ed in he a iable day), block (as andom a iable)
and day. Deg ees o eedom (d ), F- alues and signi icance le el (p) a e shown o he
ace a e and palmi a e
Publica ions and Manusc ip s
268
2.9 S udy 9: O ganic ni ogen up ake by plan s: Re-
e alua ion by posi ion-speci ic labeling o amino
acids
Daniel Mo an-Zuloaga
#1,2
, Michaela Dippold
#1,2
, B uno Glase
3
Yako Kuzyako
2,4
,
#
equal con ibu ion
1
Depa men o Ag oecosys em Resea ch, BayCEER, Uni e si y o Bay eu h
2
Depa men o Ag icul u al Soil Science, Uni e si y o Gö ingen, Ge many
3
Depa men o Soil Biogeochemis y, Ins i u e o Ag icul u al and Nu i ional Science,
Ma in-Lu he Uni e si y Halle-Wi enbe g
4
Depa men o Soil Science o Tempe a e Ecosys ems, Geo g-Augus Uni e si y o
Gö ingen
Co esponding Au ho :
Daniel Mo an-Zuloaga
Depa men o Ag icul u al Soil Science
Geo g-Augus -Uni e si y o Goe ingen
Buesgenweg 2
37077 Goe ingen
Tel: 0541-3933546
e-mail: dmo an_z@ho mail.com
Publica ions and Manusc ip s
269
Abs ac
Cu en s udies sugges ed ha besides ino ganic ni ogen (N), many plan s a e
able o ake up o ganic N in o m o amino acids. Howe e , eliable me hods o quan i y
he up ake o in ac amino acids a e s ill missing and he ele ance o o ganic N up ake is
doub ed. We used posi ion-speci ic
14
C labeling o in es iga e he up ake o in ac amino
acids and hei ole in he N nu i ion o plan s.
Posi ion speci ically
14
C and
15
N labeled alanine, injec ed in o he hizosphe e soil,
enabled o ace he up ake o C om indi idual molecule posi ions by
Zea maize, Lu-
pinus albus
and
Cicho ium in ybus
. As a con ol, uni o mly
14
C labeled alanine, ace a e
and ino ganic
15
NH
4+
and
15
NO
3-
we e applied.
The same up ake o uni o mly
14
C labeled alanine and ace a e showed ha low mo-
lecula weigh o ganic subs ances a e aken up by oo s may occu by passi e mecha-
nisms, wi hou di e ences o N con aining and N ee o ganics. Di e ences in plan up-
ake o
14
C om indi idual posi ions in alanine molecule con i med ha soil mic oo gan-
isms spli alanine wi hin 6 h in o ans o ma ion agmen s (including mine al NH
4+
), which
we e hen aken up by plan s. Only 0.04 o 0.25% o he alanine added di ec ly in o he
hizosphe e we e aken up as in ac molecule wi h he highes up ake obse ed o lupine
– he plan adap ed o o ganic N anspo om
Rhizobia
.
Mic obial u iliza ion s ongly domina ed he a e o low molecula weigh o ganic
subs ances in soils and he majo i y o amino acid
14
C up ake by plan s was explained by
passi e up ake o mic obial ans o ma ion p oduc s. Posi ion-speci ic labeling is an inno-
a i e ool ha enables o sepa a e easily he in ac up ake om up ake o molecule
agmen s. Thus, i imp o es he quan i ica ion o in ac up ake by a oiding he up o 3-
old o e es ima ion o uni o m labeling app oaches.
Keywo ds:
Alanine; Posi ion-speci ic, dual iso ope labelling; O ganic N up ake; Chico y;
Lupine; Maize; Iso opic app oaches; Ni ogen cycle

Publica ions and Manusc ip s
270
2.9.1 In oduc ion
O e he pas cen u y, many s udies ha e emphasized he ole o dissol ed ino -
ganic ni ogen (DIN) in ecosys ems (Ma son e al., 1997; Vi ousek e al., 1997; Vi ousek
e al., 1979). Ammonium (NH
4+
) and ni a e (NO
3-
) a e he main ep esen a i es o min-
e al ni ogen. Ammonium is a educed o m o DIN and can be di ec ly u ilized by plan s
a e up ake whe eas ni a e needs o be educed i s . Ni a e educ ion demands ene gy
om plan s (Doubne o a and Rysla a, 2011; Liu e al., 2011; Tischne , 2000) leading o
addi ional CO
2
luxes h ough he plan -soil sys em (Ga ichko a and Kuzyako , 2008, ,
2010). Bo h DIN species can be los om ecosys ems: ni a e by leaching in o he g ound
wa e , deni i ica ion o N
2
O and N
2
, o educ ion o ammonium and ammonium can be
los by ola iliza ion o i e e sible ixa ion by soil mine als.
In ecosys ems wi h low a ailabili y o DIN due o slow mine aliza ion, like bo eal o
a c ic ecosys ems (Nasholm e al., 1998; Vi ousek e al., 1979), plan s may also ely on
o he N o ms such as dissol ed o ganic ni ogen (DON). This is no only a sho -ci cui in
he adi ionally assumed N nu i ion pa hways ( he mine aliza ion o NH
4+
and NO
3-
is
omi ed), bu also educes po en ial N losses om ecosys ems, e.g. by leaching.
In he pas wen y yea s, he e has been ema kable in e es in DON as a plan N
sou ce (Chapin e al., 1993; Jones e al., 2005a; Nasholm e al., 1998; Paung oo-
Lonhienne e al., 2012; Schimel and Chapin, 1996). O ganic N can be ound in many
compounds in soil om mac omolecules like p o eins (Jones e al., 2005d) o humic sub-
s ances (Szajdak e al., 2003) o low molecula weigh o ganic subs ances (LMWOS) like
amino acids (Doe e al., 2012; Jones e al., 2005c; Lipson e al., 1999; S ee e e al.,
2000), amino suga s (Robe s e al., 2007; Robe s and Jones, 2012) and nucleic acids
(Kuzyako , 1996).
Many amino acids ha e e y as cycling a es and he hal -li e o amino acid C in
soils is in he ange o ew hou s (Jones e al., 2009; Kuzyako , 1996). This as cycling is
connec ed wi h as and almos comple e up ake by mic oo ganisms (Fische e al.,
2007). Ano he s udy demons a ed ha LMWOS a a e age soil concen a ions in soil
solu ion (below 10 µmol l
-1
) we e aken up by mic oo ganisms a a a e o 82% a e 3 min
(Fische e al., 2010b), and he hal -li e o amino acids in soil solu ion anges be ween 4-
8 min (Jones e al., 2004). Due o his as u iliza ion, soil mic oo ganisms a e s onge
compe i o s o amino acids han plan s (Bie na h e al., 2008; Hodge e al., 2000; Jones
e al., 2005a; Kuzyako and Xu, 2013b), whe eas in he long- e m his N is eleased by
he mic oo ganisms and is a ailable o plan s. In con as , Chapin e al. (1993) showed
in he ea ly 90ies he p e e en ial use o o ganic N by an a c ic sedge which s a ed he
discussion abou he ele ance o amino acids as plan N sou ce.
Publica ions and Manusc ip s
271
Fu he s udies showed ha bo eal o es ege a ion ac i ely ake up amino acids,
p obably due o a lack o o he N sou ces (Delgado-Baque izo e al., 2011; Nasholm e
al., 1998). The pa allel up ake o DIN and DON is dependen on hei a ailabili y
(K anabe e e al., 2007). The e o e DON is discussed o be less ele an o ag icul u al
c ops (Jones e al., 2005a). In o de o e alua e he ele ance o DON, a compa ison
be ween LMWOS wi h ino ganic N up ake was ecommended (Glass e al., 2002; Jones
e al., 2005a; S ee e e al., 2000) especially o ag oecosys ems, whe e he ole o DON
is s ill con o e sial.
Iso ope labeling o LMWOS wi h
15
N coupled wi h
13
C o
14
C is a common ool o in-
es iga e up ake and alloca ion in plan s as well as mine aliza ion o mic obial inco po a-
ion (Thede, 2010; an Hees e al., 2005). The up ake o amino acids by plan s was
mainly in es iga ed by dual-labeling wi h
15
N and
13
C (Nasholm e al., 1998; S ee e e
al., 2000). I is aci ly assumed in his app oach ha he up ake o
13
C co esponds o he
up ake o he in ac amino acid. Howe e , dual iso ope labeling has a me hodological
sho coming leading o an o e es ima ion o in ac up ake: mic oo ganisms p oduce la-
beled agmen s om he added amino acids, and hese agmen s and mine alized N
can be aken up in pa allel (Rasmussen e al., 2010). This would con ibu e o he quan i-
ied in ac up ake by he dual iso ope labeling app oach (Sauhei l e al., 2009a). The i s
e alua ion o his o e es ima ion was pe o med by he applica ion o dual uni o mly la-
beled amino acids and compound-speci ic
13
C and
15
N analysis du ing hei oo up ake. I
was shown ha due o up ake o labeled me aboli es, bulk measu emen s caused an up
o six- old o e es ima ion o he in ac up ake (Sauhei l e al., 2009a). Howe e , com-
pound-speci ic
13
C and
15
N analysis has he disad an age o being a ime-consuming and
expensi e echnique (Sauhei l e al., 2009a).
To p o e he unce ain ies o he o iginal
13
C/
15
N app oach, Rasmussen e al.
(2010) p oposed posi ion-speci ic labeling as a po en ial ool o o e come he p oblem o
molecule spli ing. Thus, up ake as a whole molecule could be dis inguished om up ake
as pa ially deg aded amino acid agmen s i.e. deca boxyla ed agmen s (Dippold and
Kuzyako , 2013). Some ecen s udies (Dijks a e al., 2011a; Fische and Kuzyako ,
2010) clea ly showed ha posi ion-speci ic
13
C and
14
C labeling enables acing he a e
o indi idual unc ional g oups in a ious soil pools. I he up ake o amino acid C occu s
as a b oad spec a o a ious ans o ma ion p oduc s (and no as in ac amino acids),
his would s ongly educe he impo ance o N nu i ion by amino acid – om a quan i a-
i e as well as a egula i e iew conce ning N de iciency.
He e, we used he same echnique o posi ion-speci ic
14
C labeling o quan i y he
in ac up ake o amino acid. We hypo hesized ha 1) he o iginal
13
C/
15
N app oach o e -
Publica ions and Manusc ip s
272
es ima es he in ac up ake o amino acids, and 2) o ganic N up ake is aceable in em-
pe a e ecosys ems bu is o mino ele ance o he N nu i ion o ag icul u al plan s.
In o de o conside he physiological di e ences o plan unc ional ypes (Weigel
e al., 2005), we pe o med ou expe imen wi h h ee species: maize, chico y and lupine.
These species di e in hei N up ake and ans o ma ion, hei physiology and mo phol-
ogy, especially in he oo sys em: 1) he g ass maize (
Zea mays
L
.) has a ib ous oo
sys em and educes NO
3-
in oo s and shoo s (He e al., 2011), 2) he he b chico y
(
Cicho ium in ybus L.
) educes NO
3-
in oo s (Goupil e al., 1998) and has a ap oo sys-
em, whe e i can s o e N-con aining compounds o he nex yea (Ameziane e al., 1997)
and 3) he legume lupine (
Lupinus
albus L.
) educes NO
3-
in oo s (Ga ichko a and
Kuzyako , 2008) and has he abili y o educe a mosphe ic N
2
in oo nodules h ough
symbiosis wi h
Rhizobia
.
As o ganic N sou ce, we used alanine as one o he mos abundan amino acids
(Fische e al., 2010a) and ammonia and ni a e as ino ganic N sou ces. To e alua e he
p e e ence o amino acid up ake compa ed o N- ee LMWOS, we included addi ional
ea men s wi h ace a e, which has a s uc u al esemblance o alanine. I up ake o N-
LMWOS (alanine) occu s mainly by unselec i e mechanisms, i should be in a simila
ange o N- ee LMWOS (ace a e).
The aims o his s udy we e: 1) o de e mine he a e o amino acids in soil wi h a
special ocus on he plan up ake o an ini ial subs ance e sus he up ake o i s ans o -
ma ion p oduc s, 2) o assess he ecological and physiological ole o in ac up ake o
amino acids by di e en plan species and 3) o e alua e he ele ance o h ee N
sou ces (alanine, ammonium and ni a e) o N nu i ion o ag icul u al plan s.
2.9.2 Ma e ial and Me hods
2.9.2.1
Expe imen p epa a ion
Soil sampling
Soil samples we e collec ed om an ag icul u al ield si e close o Hohenpölz (Ba-
a ia, Ge many a 49.907 N, 11.152 E, 501 m.a.s.l.) ha had been long- e m cul i a ed
wi h ce eals (ba ley, whea , i icale). The soil is a loamy haplic Lu isol (FAO, 2006). Soil
was collec ed om 0-10 cm, sie ed o 2 mm and oo s we e emo ed. The physico-
chemical cha ac e is ics o he soil a e desc ibed in Table 1.
Publica ions and Manusc ip s
273
Plan and ma e ial p epa a ion
A e sie ing, soil was immedia ely illed in o ans e pipe es made o low densi y
polye hylene 30 cm in leng h and 1 cm diame e , which we e used as hizo ubes
(Bie na h e al., 2008; Kuzyako and Jones, 2006).
We used maize (
Zea mays
L), lupine (
Lupinus albus
L) and chico y (
Cicho ium in-
ybus
L). Plan seeds we e p e-ge mina ed a cons an empe a u e (30 ºC) and wa e ed
o 36 hou s (Ga ichko a and Kuzyako , 2008). Then, one sp ou o each plan was in-
se ed in o he hizo ubes. The hizo ubes we e subme ged in a plas ic con aine hal -
illed wi h cold wa e o main ain he soil empe a u e a ound 12ºC. Thus, mic obial ac i -
i y e.g. mine aliza ion a es should esemble ield condi ions (Jones, 1999). The pipe e
was connec ed wi h an ai inle ( ube) a he bo om and di ec ly unde he soil su ace
(Bie na h e al., 2008) o a oid wa e sa u a ion o he soil and p o ide he soil and oo s
wi h ai .
Table 1 The physicochemical p ope ies o he Ap-ho izon o he haplic Lu isol.
Soil pa ame e s Values
pH KCl 4.88 ± 0.12
pH H
2
O 6.49 ± 0.11
To al O ganic Ca bon 1.77 ± 0.07%
To al Ni ogen 0.19 ± 0.01%
Ca ion-Exchange Capaci y 13.6 cmol
c
kg
-1
soil
Mic obial biomass C 42.5 ± 1.1 µmol C g
-1
soil
Mic obial C/N a io 9.9 ± 0.3
Chemicals and adiochemicals
The adiochemical s ock solu ion had concen a ions o 50 µM o alanine and ace-
a e, bo h wi h 10
6
DPM ml
-1
14
C ac i i y. Posi ion-speci ic labeled alanine ([1-
14
C], [2-
14
C],
[3-
14
C]alanine, Ame ican Radiolabeled Chemical Inc., S Louis, USA), as well as uni-
o mly labeled [U-
14
C]ace a e (Bio end Köln, Ge many) and [U-
14
C]alanine (Ame ican
Radiolabeled Chemical Inc., S Louis, USA) we e used.
Ni ogen labeling was pe o med wi h a 99 a om-%
15
N en iched ace o ei he
alanine CH
3
CH(
15
NH
2
)COOH as he o ganic N-sou ce o ammonium sul a e (
15
NH
4
)
2
SO
4
o po assium ni a e K
15
NO
3
as ino ganic N o ms (Bio end Köln, Ge many). Amoun o
applied C and N was iden ical in each ea men and lowe han a e age concen a ions
o alanine, ace a e, NH
4+
o NO
3-
in ag icul u al soils.
Publica ions and Manusc ip s
280
Plan species had no signi ican e ec on he amoun o mine alized
14
C (Figu e.
supplemen a y). Alanine showed signi ican ly highe mine aliza ion o C-1 (76%) han C-2
(45%) and C-3 (52%).
In gene al, we obse ed ha a e 6 h, he indi idual molecule posi ions o alanine
had s ongly di e ing a es conce ning plan up ake as well as he p opo ions emaining
in he soil.
2.9.3.4
In ac up ake o alanine assessed by posi ion-speci ic labeling
The
14
C/
15
N a io in he plan biomass (shoo s and oo s) e lec s he p opo ion o
14
C o each indi idual posi ion, which was aken up oge he wi h
15
N. Based on posi ion-
speci ic
14
C labeling, his calcula ion can be pe o med o each C posi ion o alanine (Fig.
4). This a io showed he pa e n C-3>C-2>C-1 o each plan . We conside ed ha a
molecule o alanine could only be aken up in ac i all h ee posi ions we e inco po a ed
in o he plan . Thus, he minimum o he
14
C/
15
N a io e lec s he maximum in ac up ake
o alanine in plan s, which was he case o he
14
C/
15
N a io o C-1 posi ion. These alues
we e in a simila ange o he h ee in es iga ed plan species: 7 o 14% o he alanine-N
was aken up as in ac alanine in he o de maize<chico y<lupine (Table 3).
Fig. 4 Ra io o
14
C/
15
N o indi idual alanine C posi ions inco po a ed in plan biomass.
The alanine posi ions we e C-1 (ca boxyl g oup), C-2 (amino-bound g oup) and
C-3 (me hyl g oup). Le e s indica e signi ican di e ences (p<0.001) be ween
alanine C posi ions.

Publica ions and Manusc ip s
281
In o de o compa e he con ibu ion o he h ee applied N sou ces, we es ima ed
he ace N nu i ion budge . Compa ing he ole o alanine wi hin he h ee in es iga ed
N sou ces, in ac alanine up ake eached a maximum le el o 0.25% o N. Lupine showed
he highes N up ake in he o m o in ac alanine ollowed by chico y and maize (Table
3). The ange o plan -speci ic ele ance o in ac alanine up ake (0.04-0.25%) e lec ed
he plan -speci ic abili y o N nu i ion by o ganic sou ces.
Table 3 In ac up ake o alanine by chico y, lupine and maize and es ima ed con ibu ion
o in ac alanine up ake o o al N nu i ion o hese plan s wi h espec o he
o he N sou ces.
Chico y Lupine Maize
%
15
N up ake as in ac alanine o
o al alanine-de i ed
15
N up ake 10.21
± 3.48 13.70
± 5.19 7.20
± 4.70
% in ac alanine o he h ee in-
es iga es N sou ces (alanine+
ammonium+ ni a e)
0.07
± 0.04 0.25
± 0.12 0.04
± 0.02
Fac o o o e es ima ion o in ac
up ake based on uni o m labeling 1.47
± 0.25 1.14
± 0.08 2.81
± 1.89
The up ake o in ac alanine eached a maximum o 13.7% o he o al
15
N up ake
om alanine (Table 3). The majo i y o he alanine molecules we e me abolized wi hin
6 h, when he ini ially o ganic-bound N was aken up as mine alized ammonium o e en
al eady oxidized o ni a e. This deg ada ion o alanine as a pe cen age o he applied
alanine is illus a ed in Fig. 5. In ac alanine as well as mine alized alanine-de i ed N up-
ake was highes o lupine. Once agmen ed, he up ake o C-1 was only hal o ha o
C-3. This co esponds o he highes decomposi ion o C-1. This di e en a e o indi id-
ual molecule posi ions demons a es spli ing o LMWOS which may ha e occu ed in
plan o soil.
Howe e , less han 1% o he alanine C was eco e ed in plan s a all, and he ma-
jo i y o he alanine (~99%) emains in soil o mic obial biomass. F om he alanine ag-
men s, 1.1% o 9.2% o he mine alized N was aken up by plan s, whe eas C inco po a-
ion in plan s anged only om 0.01 o 1.58% (Fig. 5). Consequen ly, only a small po ion
o he applied
14
C bu a ela i ely highe po ion o he applied
15
N was aken up by plan s
and inco po a ed in o hei biomass a e 6 h.
Publica ions and Manusc ip s
282
Fig. 5 Illus a ion o he a e o alanine ace molecules, which a e ei he aken up in-
ac o deg aded/mine alized o agmen s and subsequen ly inco po a ed in o
plan biomass o mic oo ganisms. Mic obial me abolism o alanine by mic oo -
ganisms is adap ed om Dippold & Kuzyako (in p ess)
2.9.4 Discussion
2.9.4.1
Plan up ake o N-con aining and N- ee o ganic subs ances
Ou esul s showed no p e e en ial up ake o
14
C om N-LMWOS alanine compa ed
o
14
C om ace a e – ei he aken up in ac o as agmen s - o any o he in es iga ed
plan s. Bie na h e al. (2008) ound ha maize had e en highe up ake o ace a e han
alanine. This high up ake o ace a e could mainly be a ibu ed o passi e up ake mecha-
nisms (Rasmussen e al., 2010). As shown by Jones e al. (2005c) and Ge e al. (2009),
a highe concen a ion o LMWOS and well-de eloped oo sys ems inc eases plan
compe i i eness o LMWOS compa ed o mic oo ganisms (Kuzyako and Xu, 2013a; Xu
e al., 2011). S a ing a passi e up ake means in his case he passi e, unspeci ic ans-
po o all LMWOS wi h he wa e lux owa ds he oo su ace, wi hou any di ec oo -
Publica ions and Manusc ip s
283
speci ic egula ion o amino acid anspo . Speci ic conclusions abou he con ibu ion o
a ious up ake sys ems a he plan su ace canno be s a ed om his s udy. I up ake is
domina ed by his passi e low o LMWOS owa ds he oo su ace, he bioa ailabili y o
alanine and ace a e o plan s would be he main d i e o hei up ake. Alanine can
s ongly in e ac wi h he soil ma ix by i s amino g oup, whe eas ace a e is less e ained
and consequen ly be e a ailable in he soil solu ion. In addi ion, mic oo ganisms p e e
alanine o ace a e as a subs a e (Fische e al., 2010b; an Hees e al., 2002). We did
no obse e as e decomposi ion o ace a e han alanine, bu did no quan i y inco po a-
ion in o mic obial biomass in his s udy. Alanine may ha e been p e e en ially inco po-
a ed by mic oo ganisms, as p e iously obse ed by Fische e al. (2010b), i he concen-
a ion o ee alanine in he soil solu ion was lowe han ha o ace a e.
The combina ion o high oo de elopmen and highe a ailabili y o ace a e ex-
plains he highe up ake o ace a e by maize unde he dominance o passi e lux o
LMWOS owa ds he oo s. Thus, a po en ial explana ion om hese esul s is ha
LMWOS a e aken up by plan s passi ely, i espec i e whe he hey con ain N o no .
2.9.4.2
Fa e o unc ional g oups o alanine in soil
The loss o he ca boxyl g oup by mine aliza ion is highe han ha o he me hyl
g oup in soil. Simila esul s we e shown by Fische & Kuzyako (2010) o ace a e,
Nasholm e al. (2001) o glycine, Dijks a e al. (2011a) o py u a e, and Dippold &
Kuzyako (in p ess) o alanine. In all o hese s udies based on posi ion-speci ic labeling,
he mine aliza ion o he ca boxyl g oup was as es compa ed o all o he unc ional
g oups.
Compa ing emaining alanine-
14
C in soil a e 6 h e ealed he highes mine aliza-
ion o C-1 wi h 68-70% ollowed by 30-45% and 34-52% o C-2 and C-3, espec i ely.
This decomposi ion was e en highe han ha obse ed o 3 days in a ield expe imen
being 89%, 49% and 29% o C-1, C-2 and C-3, espec i ely (Apos el e al., 2013). This
highe mine aliza ion e lec s highe mic obial ac i i y unde hizosphe e condi ions com-
pa ed o oo - ee soil (Blagoda skaya e al., 2009).
The C-1 posi ion is apidly oxidized by deca boxyla ion o he C-1 g oup o py u-
a e, he mos abundan mic obial ans o ma ion p oduc o alanine, wi hin he mic obial
me abolism. This is an ex emely as p ocess in soil (Dippold and Kuzyako , in p ess)
and kine ics o mic obial up ake and me aboliza ion a e known o be as e han plan
up ake (Jones e al., 2005a). In con as , me hyl g oups ep esen educed C and do no
need o be u he educed o many anabolic pa hways (Apos el e al., 2013; Dijks a e
al., 2011b). The e o e, C-3 was less mine alized, p e e en ially inco po a ed in o mic obial
Publica ions and Manusc ip s
284
me aboli es, also in o hose me aboli es eleased by mic oo ganisms in o soil solu ion.. In
addi ion, Dippold and Kuzyako (2013) ound a pa ial ex acellula oxida ion o alanine
ollowing he o de C-1>C-2>C-3, which may also con ibu e o a highe amoun o C-3-
agmen s in soil. These agmen s, i aken up by plan s h ough passi e mechanisms,
cause he p e e en ial inco po a ion o he C-3 posi ion (Fig. 3 and Fig. 4). This p e e en-
ial C-3 up ake as mic obial me aboli es seems o p edomina e he da k- ixa ion in he
oo s o mic obially espi ed CO
2
(which would consequen ly ha e a C-1 en ichmen ).
In summa y, mic obial up ake and u iliza ion we e he main p ocesses a ec ing he
a e o indi idual C posi ions o LMWOS in soil. P e e en ial oxida ion o C-1 and p e e -
en ial inco po a ion o C-3 by mic oo ganisms a e likely o explain he p e e en ial loss o
C-1 and accumula ion o C-3 in he en i e plan -soil sys em.
2.9.4.3
Alloca ion and ans o ma ion o C and N wi hin plan s
The p e e ence o c op plan s o NO
3-
up ake has been epo ed in many s udies
(Ge e al., 2008; He mans e al., 2006; Jones e al., 2005a) and was also con i med in
his expe imen . When ni a e was used as he N sou ce, maize educed NO
3-
in shoo s
and in oo s (Ga ichko a and Kuzyako , 2008); lupine as well as chico y educed NO
3-
mainly in oo s (Ga ichko a and Kuzyako , 2008; Goupil e al., 1998; Pa e e al., 1981).
Ou esul s co obo a e hese indings, as he highes NO
3-
anspo in o shoo s could be
ound in maize wi h a
15
N shoo / oo a io o 2.42 and lowe a ios in chico y (1.35) and
lupine (0.57) (Table 2). Low N alloca ion in o shoo s o chico y was also ound by
Ameziane e al. (1997): 8 days a e labeling, chico y kep he majo i y o
15
N in i s oo s
(shoo / oo a io 0.3). Reduced N sou ces like NH
4+
o alanine showed no clea p e e -
ence o alloca ion om oo o shoo .
S enne s am e al. (2007) ound ha inco po a ion o amino acids a e in ac up-
ake occu ed as in ac molecules, bu could no p o e his assump ion as hey nei he
labeled posi ion-speci ic no used compound-speci ic iso ope analysis (CSIA) o measu e
plan amino acids. I in ensi e me aboliza ion o amino acids in plan s occu ed, his
would lead o a p e e en ial deca boxyla ion and loss o C-1 and p e e en ial inco po a ion
o C-3, as obse ed in ou s udy. Consequen ly, his app oach would lead o an unde es-
ima ion o in ac up ake.
The e a e no many s udies in es iga ing he ans o ma ion o amino acids aken
up by plan s by means o CSIA. The in ac inco po a ion wi hou u he ans o ma ion
was i s shown by Pe sson and Nasholm (2001) by GC-MS. Sauhei l e al. (2009a), who
pe o med simila expe imen s wi h GC-C-IRMS, also ound no indica ion o oxida ion o
inco po a ed amino acids wi hin he plan me abolism. Bo h s udies excluded ans o ma-
Publica ions and Manusc ip s
285
ion o he C backbone o amino acids in o o he amino acids, bu no in o o he me abolic
p oduc s. To al
14
C and
15
N up ake e lec ed ha only mino po ion o he amino acids is
aken up and consequen ly could be me abolized by plan s. Howe e , he e is a emain-
ing unce ain y o he e ec o plan me abolism, which may con ibu e o an unde es ima-
ion o he calcula ed in ac up ake i p e e en ial C-1 oxida ion occu ed in plan s.
Posi ion-speci ic labeling in his expe imen p o ided he i s in o ma ion abou
plan ans o ma ion o alanine by compa ing he a e o indi idual molecule posi ions
wi hin he plan compa men s. In lupine (Fig. 2), posi ion C-1 was p e e en ially kep in
he oo and om posi ion C-1 o C-3, an inc easing alloca ion om oo o shoo could be
obse ed. Hence, ei he di e en agmen s o alanine we e alloca ed di e en ly wi hin
he plan o in ac alanine was pa ially spli du ing 6 h by he plan me abolism. Ge e al.
(2008) and Wa en e al. (2012) ound ha amino acids can be ans o med o o he
compounds o be anspo ed o shoo s. Howe e , Wa en e al (2012) and Sauhei l e al.
(2009a) also indica e ha ansamina ions a e he mos likely me abolic ans o ma ion
wi hin plan s and ha oxida ion o he C skele on is less likely.
The
14
C up ake by chico y was oo low compa ed o a ia ions be ween epe i ions
o de ec a compa able posi ion-speci ic end. Maize showed inc easing amoun s o
14
C
om alanine om C-1 o C-3 o shoo s and oo s. This could ei he esul om a p e e -
en ial oxida ion o C-1 and C-2 a e in ac up ake o om a p e e ed up ake o C-3 ag-
men s and hei alloca ion in o he shoo s wi hou ans o ma ion.
In summa y, ou esul s show ha e en i in ac up ake occu s, plan s end o ans-
o m LMWOS a he quickly in hei me abolism (Wegene e al., 2010) bu mainly by
ansamina ion (Sauhei l e al., 2009a). The molecula na u e o he newly o med me-
aboli es can only be cla i ied by CSIA o he ans o ma ion p oduc s.
2.9.4.4
In ac up ake o alanine in plan s
Physiological abili y o in ac alanine up ake by plan s was shown by S enne s am
e al. (2007) who iden i ied lysine his idine anspo e 1 (LHT1) as a acili a o o amino
acid up ake (lysine, glycine and alanine) by he oo s o
A abidopsis haliana
. Many s ud-
ies e alua ed he ele ance o N nu i ion by in ac amino acid up ake unde na u al soil
condi ions by using dual-iso ope bu uni o mly labeled
13
C- and
15
N- ace s (Ba dge e
al., 2003; Nasholm and Pe sson, 2001; Weigel e al., 2003). Calcula ing he
14
C/
15
N a io
o plan up ake (Fig. 4) is based on his app oach (Nasholm e al., 1998) and e lec s he
in ac alanine up ake. I we would a e age ou alanine C posi ions, which co espond o
he uni o m labeling app oach, we would de ec in ac up ake o a ound 15-18% o
alanine-de i ed N wi hou species-speci ic di e ences (Fig. 4). Calcula ing he up ake o

Publica ions and Manusc ip s
286
in ac alanine based on he C-1 posi ion, i.e. he posi ion wi h he lowes up ake, gi es
alues o in ac up ake o 7-14% o alanine-de i ed N. This demons a es a 1.2 o 3- old
o e es ima ion, i he calcula ion is based on uni o m labeling esul s compa ed o posi-
ion-speci ic labeling.
In gene al, da a o he highes in ac up ake we e ound in bo eal o es s. This was
pa ly explained by hei nu i ion ia ec o-myco hiza ion. Howe e , many s udies wi h
g assland species and annual he bs also showed highe in ac amino acid up ake han
hose obse ed in his s udy. This is a esul o he me hodological sho comings o he
uni o m
13
C o
14
C labeling app oaches. The use o posi ion-speci ic labeling enables us
o dis inguish agmen up ake om whole molecule up ake and consequen ly demon-
s a es much lowe up ake.
Rasmussen e al. (2010) expec ed he highes plan up ake o he C-1 posi ion.
They pos ula ed ha he high mine aliza ion o C-1 leads o an inc ease in HCO
3-
om C-
1 in he soil solu ion, which can be passi ely aken up by plan s (Demidchik and
Maa huis, 2007). Ou esul s con adic his concep as we obse ed he highes inco po-
a ion a e wi h C-3. Thus, i espec i e o he soil pH, a as exchange o mine alized
H
14
CO
3-
wi h a mosphe ic CO
2
leads o as
14
C losses om mine alized molecule posi-
ions. The highes up ake o C-3 suppo s he idea o plan up ake o molecule agmen s,
i.e. mic obial ans o ma ion p oduc s, by passi e up ake mechanisms.
In con as , posi ion-speci ic C-2-labeling e ealed ha ~20% o he glycine-de i ed
N was aken up as he in ac amino acid by
T i icum aes i um
(Nasholm e al., 2001).
Howe e , C-2 o glycine as a me hyl g oup esembles C-3 o alanine which had he high-
es up ake. This sugges s ha labeling o educed C posi ions (Nasholm e al., 2001) is
likely o cause an o e es ima ion o he eal in ac amino acid up ake. In addi ion, in ac
up ake o glycine may be acili a ed compa ed o alanine due o dec eased compe i i e-
ness o soil mic oo ganisms o glycine (Hocking and Je e y, 2004). In addi ion, i s
smalle molecula weigh acili a es passi e up ake. The applied amino acid concen a ion
can be ano he aspec o explain he highe ange o in ac up ake obse ed in many p e-
ious s udies. Fo example, Nasholm e al. (2001) applied a 1 mM ace solu ion,
whe eas we used a much lowe concen a ion o 50 µM. An inc eased amino acid con-
cen a ion imp o es plan compe i i eness due o ea ly sa u a ion o mic obial amino acid
anspo e s (Kuzyako and Xu, 2013a). Thus, amino acid up ake quan i ied a high con-
cen a ions may no esemble na u al condi ions as ee amino acid concen a ions a ely
exceed 100 µM in soils (Jones and Wille , 2006) and bioa ailable amino acid concen a-
ions a e e en lowe (Hobbie and Hobbie, 2012).
A e glycine applica ion o
Plan ago lanceola a
, Sauhei l e al. (2009a) quan i ied
in ac up ake a ound 16.5% o glycine-de i ed N using
13
C- and
15
N-CSIA o amino acids.
Publica ions and Manusc ip s
287
This pe cen age is sligh ly abo e he alues quan i ied he e by posi ion-speci ic
14
C label-
ing bu up o 6 old lowe han alues gained by bulk iso ope analysis. This con i ms he
o e es ima ion o in ac up ake gained by uni o mly-labeling wi h bulk iso ope analysis
app oaches.
We also ound signi ican species-speci ic di e ences in he p opo ion o in ac
alanine-
15
N o mine alized alanine-
15
N up ake (Table 3). Lupine had he highes up ake o
in ac alanine ollowed by chico y and maize (Table 3). Maize is known o ake up ei he
amino acids o hei deg ada ion agmen s (Adamczyk e al., 2012; Godlewski and
Adamczyk, 2007). The highes posi ion-speci ic di e ences measu ed in ou s udy e-
eals ha mainly mic obially ans o med C-3 agmen s o alanine we e aken up.
In con as , lupine had a high o al inco po a ion o alanine-de i ed N as well as
high up ake o in ac alanine. This can be a ibu ed o i s clus e oo s (Hawkins e al.,
2005) and o he e y e icien amino acid anspo sys ems, cha ac e is ic o legumes o
acili a e ans e om he nodules o hizobia (Day e al., 2001). Thus, plan ecophysi-
ological cha ac e is ics can inc ease hei chances o gain o ganic N.
In summa y, he use o posi ion-speci ic
13
C o
14
C labeling imp o ed he quan i ica-
ion o in ac up ake o amino acids by plan s by e ealing he con ibu ion o agmen
up ake. The highly e icien mic obial compe i ion o alanine dec eases he in ac up ake
by plan oo s.
2.9.4.5
Rele ance o amino acids as a N sou ce o ag icul u al plan s
Wi hin he h ee applied N sou ces, ni a e was p e e ed by he h ee plan s i e-
spec i e o hei ecophysiology. This p e e ence o c ops o ni a e has been shown in
p e ious s udies (Ga ichko a and Kuzyako , 2008; Ge e al., 2009; Glass e al., 2002;
Jam ga d e al., 2008) and is consis en wi h he soil p ope ies in his s udy: The Lu isol,
de eloped om loess, con ains clay mine als (mainly illi es) which can ix NH
4+
and cause
lowe plan a ailabili y o ca ionic nu ien s. Species speci ic p e e ences o N sou ces
a e in acco dance wi h p e ious s udies in g asslands (Weigel e al., 2005): as g owing
species – in ou s udy maize – showed he highes up ake o ni a e.
The up ake o alanine-
15
N was in he same ange as ammonium-
15
N. This indica es
ha p esumably he majo i y o alanine-
15
N was e y as mine alized o and aken up as
ammonium which is con i med by esul s o a p e ious s udy wi h und a species
(Schimel and Chapin, 1996).. Thus,
15
N up ake con i ms he posi ion-speci ic
14
C esul s
(Fig. 3) ha mainly pa ially me abolized o mine alized agmen s a e aken up. Also,
o he s udies ha e demons a ed as ans o ma ion o N-con aining LMWOS: Jone
s
e
al. (2004) de e mined amino acid hal -li es o 4 o 8 min in soil solu ion. Thus, wi hin one
Publica ions and Manusc ip s
288
hou , applied amino acids a e comple ely emo ed om soil solu ion and ei he inco po-
a ed in o soil mic oo ganisms, mine alized o ammonia o i e e sibly ixed by he soil
ma ix.
Table 1 shows a small C:N- a io o he mic obial biomass (~9.9) and hus a low N
demand o he mic obial communi y. Hence, he main a e o mic oo ganisms using N-
LMWOS is he C skele on. A simila s a egy o mic oo ganisms was obse ed o P-
con aining LMWOS in P- ich soils (Spohn and Kuzyako , 2013). Thus, he majo i y o
alanine-
15
N will be mine alized, eleased as
15
NH
4+
and hen be a ailable o plan up ake.
The e o e, he N mine aliza ion ac i i y o he soil mic obial communi y is a c ucial ac o
deciding whe he he inco po a ion o amino acids N occu s in ac o mine alized.
In summa y, he majo i y o alanine-de i ed N was aken up by plan s a e mine -
aliza ion and less han 1.5% o applied alanine as in ac alanine. Thus, in ac up ake o
amino acids was he leas ele an N sou ce, con ibu ing o less han 0.25% o he o al
N nu i ion o he plan . The maximal ele ance o amino acid-based N nu i ion can be
calcula ed assuming ha all 20 p o einogenous amino acids ha e an up ake simila o
alanine (al hough some o hem ha e much lowe concen a ions in he soil han alanine).
Thus, mul iplying he alanine up ake wi h 20 gi es an es ima e o he o al amino acid
up ake. Compa ing his wi h he ammonium and ni a e up ake measu ed in his s udy
e ealed ha a maximum o 5% o plan N nu i ion can be expec ed om all amino acids.
2.9.5 Conclusions and Ou look
This s udy emphasizes ha posi ion-speci ic labeling is a no el and unique ech-
nique o gain de ailed insigh in o he impo ance o o ganic N sou ces and he up ake o
LMWOS by oo s om soil. The p ecision o p e ious es ima es o in ac up ake can be
s ongly enhanced using his new labeling app oach wi hou pe o ming ime- and cos -
consuming measu emen s like compound-speci ic iso ope
13
C/
15
N analyses.
The compa ison o N-LMWOS e sus N- ee LMWOS up ake e ealed no signi i-
can di e ences in he
14
C inco po a ion om hese sou ces. This suppo ed he concep
o passi e up ake as one o he main up ake mechanisms o LMWOS by plan s.
Posi ion-speci ic
14
C labeling e ealed ha a mino po ion o amino acids was
aken up in ac , whe eas he majo i y o alanine was deg aded by soil mic oo ganisms.
Some unce ain ies emain as plan me aboliza ion like oo da k ixa ion (leading o an
o e es ima ion o in ac up ake) and plan espi a ion (leading o an unde es ima ion o
in ac up ake) canno be quan i ied by his app oach, oo.
Mine alized N as well as agmen s o he C skele on was pa ially a ailable in he
soil solu ion o oo up ake. Lupine, as he ep esen a i e o he legumes in his s udy,
Publica ions and Manusc ip s
289
con i med he gene al end o a g ea e p e e ence o legumes o o ganic N sou ces
compa ed o non-legumes which migh be a ibu ed o hei ecophysiological capabili y
o amino acid ans e be ween nodules and oo s. Maize, a plan species wi h as
g ow h, high N demand and wa e up ake showed a highe con ibu ion o passi e up ake
and hus up ake o mic obial ans o ma ion p oduc s (
14
C- agmen and DIN).
In summa y, compa ing he ele ance o DIN and amino acids o each o he in es-
iga ed plan s, i espec i e o hei ecophysiological speci ics, he ole o in ac amino acid
up ake wi hin N nu i ion was a he low. Ou s udy sugges s N up ake om o ganic
sou ces is o mino impo ance o N nu i ion o ag icul u al plan s. Ne e heless, he
ecophysiological ole canno be ully unde s ood as long as he up ake and alloca ion
mechanisms (passi e/ac i e anspo , me aboliza ion wi hin he plan ) as well as hei
egula ing ac o s a e no iden i ied. The e o e, in es iga ions wi h a b oad spec um o
posi ion-speci ic labeled LMWOS coupled wi h CSIA o plan and mic obial ans o ma ion
p oduc s a e needed.
Acknowledgemen s
This s udy was inanced by Deu sche Fo schungsgemeinscha DFG. We hank Ilse
Thau elde and S e anie Bösel, echnical s a a he Uni e si y o Bay eu h and Ma in-
Lu he Uni e si y o Halle, espec i ely, C. Ca edon o igu e design and C. We ne o
he ho ough e iew.

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h p://dx.doi.o g/10.1016/j.soilbio.2013.05.021
Bi k, Jago Jona han, Dippold M, Wiesenbe g GLB, Glase B (2012): Combined quan i i-
ca ion o aecal s e ols, s anols, s anones and bile acids in soils and e es ial
sedimen s by gas ch oma og aphy-mass spec ome y. Jou nal o Ch oma og a-
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Glase , B uno, Benesch M, Dippold M, Zech W. (2012): In si u N-15 and C-13 labelling o
indigenous and plan a ion ee species in a opical moun ain o es (Munessa,
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Zech, Wol gang, Zech R, Zech M, Leibe K, Dippold M, F echen M, Busse R, And ee A
(2011): Obliqui y o cing o Qua e na y glacia ion and en i onmen al changes in
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Acknowledgemen s
A-2
Acknowledgemen s
Ich danke allen, die mi wäh end meine Dok o a bei zu Sei e s anden ganz he z-
lich.
Mein besonde e Dank gil meinem Dok o a e P o . D . Yako Kuzyako : du ch
einem au mein In e essengebie zugeschni enen DFG An ag e möglich e e mi die
P omo ion in diesem spannenden Fo schungs eld zwischen Biochemie und Bodenkunde.
E lies mi s e s den nö igen F ei aum eigene Ideen in allen Ebenen wissenscha lich A -
bei ens zu e wi klichen, was mich auße o den lich mo i ie e. Auch ü die in ensi e Ein-
üh ung in die Wissenscha sgemeinde im Rahmen zahl eiche Tagungs eilnahmen
möch e ich mich bei ihm ech he zlich bedanken.
Ein auße o den liche Dank geh an P o . D . B uno Glase an dessen Ins i u e ein
maßgebliche An eil de Analy ik e olg e. Aus den zahl eichen Diskussionen und Ge-
sp ächen konn e ich sowohl wissenscha lich als auch pe sönlich seh p o i ie en. Im
speziellen ü seine F eundscha und sein imme o enes Oh möch e ich mich ech
he zlich bedanken.
Des Wei e en gil mein Dank P o . Huwe, de wäh end meine Dok o a bei Labo -
äume und Ge ä scha en zu Fe igs ellng meine Disse a ion be ei s ell e. Ebensog o-
ße Dank gil P o . D . Ch is iane We ne Pin o, die mich seh he zlich in ih e A bei s-
g uppe au nahm und mi in ielen Gesp ächen s e s hil eich zu Sei e s and. Insbeson-
de e möch e ich P o . D . Wol gang Zech danken, du ch dessen Fö de ung ich ü die
Bodenkunde begeis e wu de und au dessen Un e s ü zung ich imme zu ückg ei en
konn e.
De DFG danke ich ü die Finanzie ung dieses P ojek s (DFG KU 1184/19-1) zu
Au klä ung posi ionsspezi ische T ans o ma ionen in Böden.
Ein besonde es Dankeschön geh an S e anie Bösel, de en Geschick am Iso o-
penmassenspek ome e wich igs e Vo ausse zung ü das Zus andekommen des Um-
ang eichen Da ensa zes wa . Die aus den ielen gemeinsamen S unden am IRMS en -
s andene F eudscha ha mi im Rahmen meine Disse a ion seh gehol en. Auch Ilse
Thau elde , die imme mi Ra und Ta zu Sei e s and, ha meine Labo a bei in Bay eu h
seh e leich e und be eiche .
Spezielle Dank gil meinen beiden Diploma bei sbe eue n D . Leopold Sauhei l
und Jago Bi k, denen ich das Wissen und die analy ischen Fähigkei en zu Du ch üh ung
diese Dok o a bei e danke. Neben eine o lau end, kons uk i en Zusammena bei
mi beiden s anden sie mi auch als F eunde in jede Lebenslage zu Sei e. Ebenso in-
Acknowledgemen s
A-3
ensi e Un e s ü zung habe ich du ch PD D . Michael Zech e ah en, de sowohl achlich
in ielen Diskussionen zum Gelingen de Disse a ion beige agen ha als auch pe sön-
lich mein Leben seh be eiche ha . Fü die kons uk i e Zusammena bei und ih e s e s
hil eiche F eundscha möch e ich mich auße dem bei D . Guido Wiesenbe g und D .
Bjö n Buggle bedanken.
Tie e Dankba kei emp inde ich ü meine beiden, euen F eundinnen Janine
Somme und Ka ha ina Leibe , de en F eundscha ich mi wäh end all de Jah e unab-
hängig on allen äuße en Fak o en imme siche sein konn e. Neben de ak i en Un e -
s ü zung in Feld und Labo ha .a. die K a , die ich aus diesen F eundscha en ziehen
konn e, das Zus andekommen diese Dok o a bei e möglich .
Meinen he zlichen Dank möch e ich allen Ko ek u lese n diese A bei ausp echen,
Leopold Sauhei l, B uno Glase , Michael Zech, Thomas F iedel, Ca olin Apos el und
meine Schwes e Ch is ine.
Ein in Wo e nich zu assende Dank geh geh jedoch an meine Familie – meine
Schwes e Ch is ine, meinem B ude Tobias, meinem Va e Klemens, Ge linde, und mei-
ne Pa in Anna, die allen näch lichen Labo schich en, s ändigen Diens eisen und age-
langen Compu e sessions zum T o z mich imme un e s ü z haben. Ih e Liebe und Rü-
ckendeckung in jede Lebenslage wa en die G undlage ü das Zus andekommen diese
A bei .
(Eidess a liche) Ve siche ungen und E klä ung
A-4
(Eidess a liche) Ve siche ungen und E klä ungen
(§ 5 N . 4 P omO)
Hie mi e klä e ich, dass keine Ta sachen o liegen, die mich nach den gese zlichen Be-
s immungen übe die Füh ung akademische G ade zu Füh ung eines Dok o g ades
unwü dig e scheinen lassen.
(§ 8 S. 2 N . 5 P omO)
Hie mi e klä e ich mich dami ein e s anden, dass die elek onische Fassung meine
Disse a ion un e Wah ung meine U hebe ech e und des Da enschu zes eine geson-
de en Übe p ü ung hinsich lich de eigens ändigen An e igung de Disse a ion un e zo-
gen we den kann.
(§ 8 S. 2 N . 7 P omO)
Hie mi e klä e ich eidess a lich, dass ich die Disse a ion selbs s ändig e ass und kei-
ne ande en als die on mi angegebenen Quellen und Hil smi el benu z habe.
Ich habe die Disse a ion nich be ei s zu E langung eines akademischen G ades an-
de wei ig einge eich und habe auch nich be ei s diese ode eine gleicha ige Dok o p ü -
ung endgül ig nich bes anden.
(§ 8 S. 2 N . 9 P omO)
Hie m e klä e ich, dass ich keine Hil e on gewe blichen P omo ionsbe a e n bzw.
- e mi le n in Ansp uch genommen habe und auch kün ig nich nehmen we de.
Bay eu h, 31.03.2014
O , Da um, Un e sch i