Metabolic Pathways of Amino Acids, Monosaccharides and Organic Acids in Soils assessed by ...

324 Pages·2014·52.09 MB·English

Metabolic Pathways of
Amino Acids, Monosaccharides and Organic Acids
in Soils
assessed by Position-Specific Labeling

Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften
(Dr. rer. nat.)
an der Fakultät Biologie / Chemie / Geowissenschaften
der Universität Bayreuth

vorgelegt von
Michaela A. Dippold
(Dipl. Geoökologin & Dipl. Biochemikerin)
geb. am 10.07.1982 in Bamberg

Betreuer: Prof. Dr. Yakov Kuzyakov

Bayreuth, den 11. September 2013
I
Die vorliegende Arbeit wurde in der Zeit von 01.03.2010 bis 11.10.2013
in Bayreuth am Lehrstuhl für Agrarökosystemforschung unter der
Betreuung von Herrn Prof. Dr. Yakov Kuzyakov angefertigt.

Vollständiger Abdruck der von der Fakultät für Biolgie, Chemie und
Geowissenschaften der Universität Bayreuth genehmigten Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwis-
senschaften (Dr. rer. nat.).

Dissertation eingereicht am: 11.09.2013
Zugelassen durch die Prüfungskommission: 18.09.2013
Wissenschaftliches Kolloquium: 31.01.2014

Amtierender Dekan: Prof. Dr. Rhett Kempe

Prüfungsauschuss:
Prof. Dr. Y. Kuzyakov (Erstgutachter)
Prof. Dr. C. Werner-Pinto (Zweitgutachter)
Prof. Dr. E. Matzner (Vorsitz)
PD Dr. M. Horn

Drittgutachter: PD Dr. G. Wiesenberg

II
Contents

I. Contents

I. Contents...............................................................................................I
II. List of Figures....................................................................................IX
III. List of Tables ...................................................................................XV
IV. Abbreviations.................................................................................XVII
V. Summary.......................................................................................XVIII
VI. Zusammenfassung..........................................................................XX
1 Extended Summary.............................................................................................. 1
1.1 Introduction...................................................................................................... 1
1.1.1 Low molecular weight organic substances in soils.......................................... 1
1.1.1.1 Role and relevance of low molecular weight organic substances in soil...1
1.1.1.2 Sources and sinks of LMWOS.................................................................2
1.1.2 Microbial utilization of low molecular weight organic substances.................... 4
1.1.2.1 Microbial uptake of LMWOS: the most competitive process determining
the fate of LMWOS-C in soil....................................................................4
1.1.2.2 Mineralization versus incorporation of LMWOS-C into microorganisms...5
1.1.3 Metabolic tracing by position-specific labeling................................................ 6
1.1.4 Objectives...................................................................................................... 8
1.2 Experiments and Methods............................................................................... 8
1.2.1 Field experiment............................................................................................. 8
1.2.2 Laboratory experiments.................................................................................10
1.2.2.1 Experiment 1: Transformations of free alanine.......................................10
1.2.2.2 Experiment 2: Transformations of sorbed alanine..................................11
1.2.2.3 Experiment 3: Plant uptake of intact alanine..........................................11
1.2.3 Methods to trace 13C and 14C in transformation products of LMWOS.............11
1.2.3.1 Bulk-isotope measurements by EA-IRMS..............................................12
1.2.3.2 Compound-specific isotope analysis of microbial biomarkers................12
1.2.3.3 Radiochemical analyses........................................................................13
1.2.4 The Divergence Index...................................................................................14
1.3 Results and Discussion..................................................................................15
1.3.1 Overview: main results of the studies............................................................15
I
Contents
1.3.2 Determination of metabolic pathways of amino acids, monosaccharides and
organic acids.................................................................................................16
1.3.2.1 Similarities and differences of individual LMWOS..................................16
1.3.2.2 The main pathways for LMWOS metabolization by soil microorganisms19
1.3.2.3 Metabolic pathways for the formation of specific cellular compounds....22
1.3.3 Identification of specific metabolic pathways..................................................24
1.3.3.1 Specific pathways of individual members of the microbial community in
soils.......................................................................................................24
1.3.3.2 Pathways under various concentrations of LMWOS..............................25
1.3.3.3 Pathways of sorbed LMWOS.................................................................27
1.3.3.4 Extra- versus intracellular transformation pathways...............................29
1.3.4 Kinetics and ecological relevance of competing sinks for LMWOS................30
1.3.4.1 Sorption versus microbial utilization.......................................................30
1.3.4.2 Plant uptake versus microbial utilization................................................30
1.4 Conclusions.....................................................................................................32
1.5 Reference List.................................................................................................35
1.6 Contribution to the included manuscripts and publications........................40
2 Publications and Manuscripts............................................................................44
2.1 Study 1: Fate of low molecular weight organic substances in an arable soil:
from microbial uptake to utilisation and stabilisation..................................44
Abstract....................................................................................................................45
2.1.1 Introduction...................................................................................................47
2.1.2 Material and Methods....................................................................................49
2.1.2.1 Experimental design..............................................................................49
2.1.2.2 Bulk soil δ13C analysis...........................................................................50
2.1.2.3 Microbial biomass..................................................................................50
2.1.2.4 Phospholipid fatty acid analysis.............................................................51
2.1.2.5 Calculations and statistical analysis.......................................................52
2.1.3 Results..........................................................................................................53
2.1.3.1 Microbial community structure...............................................................53
2.1.3.2 Microbial utilisation of LMWOS..............................................................54
2.1.3.3 Utilisation of LMWOS by functional microbial groups.............................55
2.1.4 Discussion.....................................................................................................59
2.1.4.1 Incorporation of LMWOS into SOM and microbial biomass....................59
2.1.4.2 Microbial community composition..........................................................62
2.1.4.3 Incorporation of LMWOS into PLFAs.....................................................63
II
Contents
2.1.5 Conclusion....................................................................................................66
Acknowledgements...................................................................................................67
References...............................................................................................................68
Supplementary Data.................................................................................................72
2.2 Study 2: Improved δ13C analysis of amino sugars in soil by Ion
Chromatography – Oxidation – Isotope Ratio Mass Spectrometry.............74
Abstract....................................................................................................................75
2.2.1 Introduction...................................................................................................76
2.2.2 Material and Methods....................................................................................78
2.2.2.1 Soil........................................................................................................78
2.2.2.2 Chemicals, reagents and external and internal standards......................78
2.2.2.3 Soil hydrolysis and ion removal .............................................................79
2.2.2.4 Purification by cation exchange column.................................................79
2.2.2.5 Development of the measurement by IC-O-IRMS..................................79
2.2.2.6 Evaluation of amino sugar quantification via IC-O-IRMS........................81
2.2.2.7 Evaluation of δ13C determination via IC-O-IRMS....................................82
2.2.3 Results and Discussion.................................................................................83
2.2.3.1 Chromatography....................................................................................83
2.2.3.2 Recovery, linearity, precision and detection and quantification limits.....84
2.2.3.3 3.3 Amount dependence and correction factors of δ13C values..............85
2.2.3.4 3.4 Accuracy, precision and isotopic LoQ of δ13C determination............86
2.2.3.5 Advantages of IC-O-IRMS.....................................................................89
2.2.4 Conclusions...................................................................................................89
Acknowledgments.....................................................................................................90
Reference List...........................................................................................................91
Supplementary Data.................................................................................................93
2.3 Study 3: Biochemical pathways of amino acids in soil: Evaluation by
position-specific labeling and 13C-PLFA analysis.........................................96
Abstract....................................................................................................................97
2.3.1 Introduction...................................................................................................98
2.3.2 Material and Methods..................................................................................100
2.3.2.1 Field experiment..................................................................................100
2.3.2.2 Analytical methods...............................................................................101
2.3.2.3 Divergence Index.................................................................................104
2.3.2.4 Statistical analysis...............................................................................105
III
Contents
2.3.3 Results........................................................................................................105
2.3.3.1 Incorporation of uniformly labeled amino acids....................................105
2.3.3.2 Incorporation of position-specifically labeled amino acids....................106
2.3.3.3 Divergence Index.................................................................................108
2.3.4 Discussion...................................................................................................110
2.3.4.1 Incorporation of carbon from amino acids in soil and microbial biomass
110
2.3.4.2 Incorporation of tracer into the microbial groups..................................111
2.3.4.3 Discrimination of individual carbon positions by microbial utilization differs
depending on oxidation state, amino acid and time..............................113
2.3.5 Conclusions.................................................................................................115
Acknowledgements.................................................................................................116
Reference List.........................................................................................................117
Supplementary Data...............................................................................................120
2.4 Study 4: Biogeochemical transformations of amino acids in soil assessed
by position-specific labeling........................................................................122
Abstract..................................................................................................................123
2.4.1 Introduction.................................................................................................124
2.4.2 Material and Methods..................................................................................126
2.4.2.1 Soil......................................................................................................126
2.4.2.2 Chemicals and radiochemicals............................................................126
2.4.2.3 Experimental setup..............................................................................126
2.4.2.4 Radiochemical analyses......................................................................129
2.4.2.5 Calculation of the kinetics of alanine utilization....................................129
2.4.2.6 Calculation of the distribution of alanine-C in transformation products.130
2.4.2.7 Statistics..............................................................................................131
2.4.3 Results........................................................................................................132
2.4.3.1 Evaluation of results quality.................................................................132
2.4.3.2 Sorption of alanine to the soil matrix....................................................133
2.4.3.3 Kinetics of biotic alanine utilization.......................................................134
2.4.3.4 Biotic transformation products of alanine.............................................136
2.4.3.5 Position-specific differences of the alanine transformation pathways...137
2.4.4 Discussion...................................................................................................138
2.4.4.1 Sorption of alanine occurs as a whole molecule..................................138
2.4.4.2 Kinetics of extracellular transformation and microbial uptake...............139
2.4.4.3 Exoenzymatic transformation products................................................140
IV
Contents
2.4.4.4 Metabolic pathways and their intracellular transformation products.....141
2.4.5 Conclusions and Outlook.............................................................................143
Acknowledgement..................................................................................................144
Reference List.........................................................................................................145
Supplementary Data...............................................................................................148
2.5 Study 5: Sorption affects amino acid pathways in soil: Implication from
position-specific labeling of alanine............................................................151
Abstract..................................................................................................................152
2.5.1 Introduction.................................................................................................154
2.5.2 Material and Methods..................................................................................156
2.5.2.1 Soil......................................................................................................156
2.5.2.2 Sorbents..............................................................................................156
2.5.2.3 Chemicals and radiochemicals............................................................157
2.5.2.4 Pre-experiments..................................................................................157
2.5.2.5 Experimental Setup.............................................................................158
2.5.2.6 Chemical and radiochemical analyses.................................................159
2.5.2.7 Calculations and modeling...................................................................159
2.5.2.8 Calculation of the C-1/C-2,3-ratio and the Divergence Index DI ..........160
i
2.5.2.9 Statistics..............................................................................................161
2.5.3 Results........................................................................................................162
2.5.3.1 Sorption and microbial utilization of uniformly labeled alanine.............162
2.5.3.2 Kinetics of position-specific utilization of sorbed alanine C...................164
2.5.3.3 Incorporation of C from alanine positions in stabilized pools and
decomposition to CO ..........................................................................167
2
2.5.4 Discussion...................................................................................................169
2.5.4.1 Sorption mechanisms of amino acids..................................................169
2.5.4.2 Bioavailability of sorbed alanine...........................................................172
2.5.4.3 Pathways of microbial metabolization of sorbed alanine......................173
2.5.4.4 Stabilization of amino acid C by sorption.............................................176
2.5.5 Conclusions and Outlook.............................................................................177
Acknowledgments...................................................................................................178
Reference List.........................................................................................................179
Supplementary Data...............................................................................................183
2.6 Study 6: Biochemistry of hexose and pentose transformations in soil
analyzed by position-specific labeling and 13C-PLFA.................................184
Abstract..................................................................................................................185
V
Contents
2.6.1 Introduction.................................................................................................186
2.6.2 Material and Methods..................................................................................188
2.6.2.1 Sampling Site......................................................................................188
2.6.2.2 Analytical methods...............................................................................189
2.6.2.3 Divergence Index.................................................................................192
2.6.2.4 Statistical analysis...............................................................................192
2.6.3 Results........................................................................................................193
2.6.3.1 Incorporation of uniformly labeled monosaccharides...........................193
2.6.3.2 Incorporation of position-specifically labeled monosaccharides...........193
2.6.3.3 Tracer uptake of functional microbial groups........................................195
2.6.3.4 Divergence Index.................................................................................196
2.6.4 Discussion...................................................................................................196
2.6.4.1 Glucose and Ribose incorporation into soil and microbial biomass......196
2.6.4.2 Microbial utilization of individual positions of glucose and ribose
molecules............................................................................................198
2.6.4.3 Specific pathways of glucose and ribose utilization by individual microbial
groups.................................................................................................199
2.6.4.4 Metabolic tracing by position-specific labeling of monosaccharides.....202
2.6.5 Conclusions and Outlook.............................................................................203
Acknowledgements.................................................................................................204
References.............................................................................................................205
Supplementary Data...............................................................................................208
2.7 Study 7: Metabolic pathways of fungal and bacterial amino sugar formation
in soil assessed by position-specific 13C-labeling......................................210
Abstract..................................................................................................................211
2.7.1 Introduction.................................................................................................213
2.7.2 Material and Methods..................................................................................215
2.7.2.1 Experimental Site................................................................................215
2.7.2.2 Experiment Design..............................................................................215
2.7.2.3 Sampling and Sample Preparation......................................................216
2.7.2.4 Bulk Soil and Microbial Biomass Analysis............................................216
2.7.2.5 Amino sugar δ13C analysis...................................................................217
2.7.2.6 Divergence Index.................................................................................219
2.7.2.7 Statistics..............................................................................................220
2.7.3 Results........................................................................................................220
2.7.3.1 Glucose 13C incorporation into soil and microbial C pools....................220
VI
Contents
2.7.3.2 Incorporation of C from various positions of glucose molecule into
individual amino sugars.......................................................................221
2.7.3.3 Replacement of cell wall pool by glucose 13C.......................................223
2.7.4 Discussion...................................................................................................224
2.7.4.1 Fungal versus bacterial contribution to the amino sugar fingerprint and
glucose utilization................................................................................224
2.7.4.2 Pathways of amino sugar formation.....................................................225
2.7.4.3 Specific pathways of fungi and bacteria...............................................228
2.7.5 Conclusions and Outlook.............................................................................229
Acknowledgements.................................................................................................230
References.............................................................................................................231
Supplementary Data...............................................................................................235
2.8 Study 8: Formation and transformation of fatty acids in soil assessed by
position-specific labeling of precursors......................................................236
Abstract..................................................................................................................237
2.8.1 Introduction.................................................................................................239
2.8.2 Material and Methods..................................................................................241
2.8.2.1 Experimental Site................................................................................241
2.8.2.2 Experiment Design..............................................................................242
2.8.2.3 Sampling and Sample Preparation......................................................242
2.8.2.4 Bulk Soil and Microbial Biomass Analysis............................................242
2.8.2.5 PLFA δ13C analysis..............................................................................243
2.8.2.6 Fatty acid grouping..............................................................................245
2.8.2.7 The Divergence Index DI.....................................................................245
i
2.8.2.8 Statistics..............................................................................................246
2.8.3 Results........................................................................................................246
2.8.3.1 Incorporation of 13C in soil and microbial biomass................................246
2.8.3.2 Incorporation of C from various positions of acetate and palmitate into
individual PLFAs..................................................................................247
2.8.3.3 Incorporation of acetate and palmitate 13C into PLFAs of individual
microbial groups..................................................................................249
2.8.4 Discussion...................................................................................................252
2.8.4.1 Utilization and turnover of acetate and palmitate by soil microbial
community...........................................................................................252
2.8.4.2 Pathways of fatty acid formation from acetate in soil............................253
2.8.4.3 Pathways of fatty acid transformations in soils.....................................254
VII
Contents
2.8.4.4 Pathways of specific microbial groups in soils......................................255
2.8.4.5 Consequences for the application of fatty acids as biomarkers............257
2.8.5 Conclusions and Outlook.............................................................................258
Acknowledgements.................................................................................................259
References.............................................................................................................260
Supplementary Data...............................................................................................265
2.9 Study 9: Organic nitrogen uptake by plants: Reevaluation by position-
specific labeling of amino acids...................................................................268
Abstract..................................................................................................................269
2.9.1 Introduction.................................................................................................270
2.9.2 Material and Methods..................................................................................272
2.9.2.1 Experiment preparation.......................................................................272
2.9.2.2 Experimental setup..............................................................................274
2.9.2.3 Laboratory analysis..............................................................................274
2.9.2.4 Calculation of intact uptake of labeled substances...............................275
2.9.2.5 Statistical analysis...............................................................................277
2.9.3 Results........................................................................................................277
2.9.3.1 15N uptake in plants from organic and inorganic N sources..................277
2.9.3.2 Plant uptake of uniformly 14C labeled alanine and acetate...................278
2.9.3.3 14C plant uptake of position-specific labeled alanine............................279
2.9.3.4 Intact uptake of alanine assessed by position-specific labeling............280
2.9.4 Discussion...................................................................................................282
2.9.4.1 Plant uptake of N-containing and N-free organic substances...............282
2.9.4.2 Fate of functional groups of alanine in soil...........................................283
2.9.4.3 Allocation and transformation of C and N within plants........................284
2.9.4.4 Intact uptake of alanine in plants..........................................................285
2.9.4.5 Relevance of amino acids as a N source for agricultural plants...........287
2.9.5 Conclusions and Outlook.............................................................................288
Acknowledgements.................................................................................................289
References.............................................................................................................290
Supplementary Data...............................................................................................295
Additional peer-reviewed pubications...................................................1
Acknowledgements.................................................................................2

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