Tetramethylammonium hydroxide thermochemolysis for the analysis of cellulose and free carbohydrates in a peatbog Céline Estournel-Pelardy, Frédéric Delarue, Laurent Grasset, Fatima Laggoun-Défarge, André Amblès
To cite this version: Céline Estournel-Pelardy, Frédéric Delarue, Laurent Grasset, Fatima Laggoun-Défarge, André Amblès. Tetramethylammonium hydroxide thermochemolysis for the analysis of cellulose and free carbohydrates in a peatbog. Journal of Analytical and Applied Pyrolysis, Elsevier, 2011, 92 (2), pp.401-406. �10.1016/j.jaap.2011.08.004�. �insu-00615435�
HAL Id: insu-00615435 https://hal-insu.archives-ouvertes.fr/insu-00615435 Submitted on 19 Aug 2011
HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Tetramethylammonium hydroxide thermochemolysis for the analysis of cellulose and free carbohydrates in a peatbog
Céline Estournel-Pelardya, Frédéric Delarueb, Laurent Grasseta*, Fatima Laggoun-Défargeb, André Amblèsa a Université de Poitiers, CNRS, Laboratoire de Synthèse et de Réactivité des Substances Naturelles - UMR 6514, 4 rue M. Brunet, 86022 Poitiers cedex, France. b Université d'Orléans, Université François Rabelais - Tours, CNRS/INSU. Institut des Sciences de la Terre d’Orléans - UMR 6113. Campus Géosciences. 1A rue de la Férollerie, 45071 Orléans cedex 2, France. E-mail: [email protected]
1 2 3 4 5 6 7 8 9 10 11 12 13 14
We have compared TMAH thermochemolysis with the classical method using acid hydrolysis
for carbohydrates analysis in a peat core. Even if TMAH thermochemolysis does not analyse
hemicellulosic carbohydrates and discriminate each individual carbohydrate sensu stricto, it
allows the analysis of a cellulose pool hidden to acid hydrolysis and the specific analysis of
free and terminal carbohydrates. Simple direct comparisons of thermochemolysis data with
data generated from acid hydrolysis cannot be done because of the different mechanisms
involved in each process. TMAH thermochemolysis must be viewed and used as a pertinent
and complementary method for the analysis of carbohydrates protected and trapped by the
organic matter in complex environmental systems.
24 1. Introduction
Because of their ubiquity and abundance, carbohydrates are potentially useful compounds
in elucidating sources, processes and pathways of biologically important organic materials in
Acid hydrolysis is currently used to liberate carbohydrates from soils. It involves acid
hydrolysis and purification before their analysis by liquid chromatography or by gas
chromatography after derivatization. Recommended procedures either use H2SO4 [1-6], HCl
[1, 6-8] or TFA [1, 9-10] to cleave glycosidic bonds of polysaccharides yielding sugar
monomers. These methods can be applied to a wide range of soil samples and are easy to
perform but yet time-consuming.
Tetramethylammonium hydroxide (TMAH) is the most common reagent used for
thermochemolysis (more than 90% of published thermochemolysis applications have used
Page 1 of 18
TMAH ). It is used for the analysis of complex and intractable samples such as soils and
sediments. For that kind of samples, TMAH thermochemolysis provides useful information
simultaneously on a wide range of compounds related to lipids, lignins, tannins, proteins and
carbohydrates markers. Due to its capability to cleave common ester and ether bonds and to
methylate acidic functional groups, TMAH thermochemolysis allows also the analyses of
compounds entrapped in macromolecular network . However, few studies have identified these products in soil samples [13-14]. It could be
partly due to the relative poor sensitivity of TMAH thermochemolysis to carbohydrates both
at high temperature (up to 600°C) [15-16] or with extended reaction time (250°C during 30
min in sealed tube) .
On the other hand, several studies have shown that TMAH thermochemolysis of
individual sugars releases, beside products formed by recombination of previously cleaved
fragments, 3-deoxyaldonic acids methyl esters resulting from the isomerisation of the C-2
position and dehydration of the C-3 position. These products have conserved their original
conformation at the C-4 and C-5 positions [13, 18-19]. As a consequence, aldohexoses such
as glucose, mannose and allose give identical 3-deoxyaldonic acids methyl esters but different
ones from their C-4 epimers (i.e. galactose, gulose and idose). In the same way, the 6-
deoxyhexoses, fucose and rhamnose (the C-4 epimer of fucose), give different saccharinic
acids methyl esters. For the same reason, aldopentoses (xylose, arabinose, ribose and lyxose)
give the same saccharinic acid methyl esters (Fig. 1). The methylated forms of these
saccharinic products have mass spectra with m/z 129 as base peak (for the interpretation of the
electron impact MS fragmentation of permethylated saccharinic acids see Fabbri and Helleur
(1999)  and Bleton et al. (1996) ). Furthermore, although their mass spectra present
no evident differences, they present different chromatographic behaviour with different
retention times in gas chromatography. Moreover, TMAH thermochemolysis of cellulose
produces a specific epimeric pair of methylated isosaccharinic acids producing specific ions
in EI mass spectroscopy (i.e. m/z 173) . Then, to detect monosaccharides and glycosidic
units from cellulose with TMAH thermochemolysis, single ion monitoring at m/z 129 and 173
can be used to reveal their presence in complex materials.
Soil organic matter is composed of more or less altered and inherited biochemical
hemicellulose, cellulose and microbial
sugars (i.e. mainly
exopolysaccharides). Depending on organic matter sources and degradation, carbohydrate
monomers exhibit distinctive composition patterns. Ombrotrophic peat bogs are covered
mainly by Sphagnum spp. with Eriophorum as the dominant vascular species. Cyperaceae are
Page 2 of 18
rich in xylose (one of the most abundant aldopentoses in living kingdom) [8, 21] as well as in
arabinose [22-24]. Conversely, galactose, mannose and rhamnose are adequate indicators for
mosses, in particular Sphagnum spp. [22, 25]. In addition, fucose could be considered as a
microbial marker in peat bogs . At 400°C, a recent study has shown that the identification of carbohydrates derivatives in
soil samples is possible with TMAH thermochemolysis . In addition, TMAH
thermochemolysis can be applied to precise and large quantities of material (up to 1g) since
an off-line preparative technique was developed .
Samples collected at different depths of a peat core were submitted to TMAH
Under the same thermochemolysis conditions, cellulose and the most abundant
monosaccharides encountered in peat bog were submitted to thermochemolysis allowing their
assignment among the peat samples products based both on the mass spectra and on the
retention times in gas chromatography of their thermochemolysis derivatives. Despite
unavoidable matrix effects, this off-line preparative thermochemolysis also allows a semi-
quantification of thermochemolysis products when known quantities of standards were
analysed in the same conditions than the studied complex samples.
Results were compared with those obtained using a classical method for carbohydrates
analyses (acid hydrolysis with H2SO4) to have insight on the capability of TMAH
thermochemolysis for the carbohydrate analyses in soils and sediments.
94 95 96
2. Materials and methods
Aldopentoses (D-xylose, L-arabinose, D-ribose), aldohexoses (D-glucose, D-mannose, D-
galactose), deoxyhexoses (L-rhamnose, L-fucose), cellulose, deoxy-6-glucose, H2SO4, CaCO3
CH3OH, CH2Cl2, LiClO4 and TMAH were purchased from Sigma-Aldrich (St Louis, USA)
and BSTFA+TMCS (99:1) from Supelco (Bellefonte, USA).
2.2. Peat samples
Page 3 of 18
Samples were collected in June 2008 at the open bog part of an undisturbed Sphagnum-
dominated mire in the Jura Mountains (Le Forbonnet peatland, France), which has been
described in detail . The site is protected by the EU Habitat Directive of Natura 2000 and
has been classified as a Region Natural Reserve for more than 20 yr. Annual precipitation is
about 1300–1500 mm per year, with a mean annual air temperature of 7–8 °C. Samples were
collected at different depths: (i) two samples were collected in the upper oxic part (acrotelm),
(ii) two in the water table zone (mesotelm) and (iii) two in the anoxic part (catotelm) (Fig. 2).
They were air dried (40°C), finely ground and stored at -20°C before further analysis.
2.3. Carbohydrate analysis by hot water extraction and H2SO4 hydrolysis
The detailed H2SO4 hydrolysis method has been described elsewhere . The analysis is
carried out in two experiments. The first operating procedure for total sugar analysis can be
summarized as follows: 1 ml of H2SO4 (12M) was added to 20 to 30 mg of dry peat in a
Pyrex® test tube. After 16 h at room temperature, the samples were diluted with 9 ml of H2O
to a 1.2 M concentration in H2SO4. The tube was tightly closed under vacuum and heated at
100°C for 4 h. After cooling, deoxy-6-glucose (0.4 mg.ml-1 in water) was added as internal
standard . The sample was subsequently neutralised with CaCO3. The precipitate was
removed by centrifugation and the supernatant evaporated to dryness. The sugars were then
dissolved in CH3OH and the solution purified by centrifugation. After transferring the
solution to another vessel, the solvent was evaporated under vacuum. The sugars were
dissolved in pyridine containing 1 % (v/v) of LiClO4 and left 16 h at 60°C for anomer
equilibration . In both cases, they were silylated by BSTFA+TMCS (99:1) and analysed
using a Perkin–Elmer AutoSystem XL GC (split injector, 240°C; flame ionization detector
(FID), 300°C) with a fused silica capillary column (CPSil5CB, 25 m length, 0.25 mm i.d.,
0.25 µm film thickness) and helium as carrier gas. The GC was temperature programmed
from 60 to 120°C at 30°C.min-1 (isothermal for 1 min) and raised to 240°C at 5°C.min-1 and
finally at 20°C.min-1 to 310°C and maintained at that temperature for 10 min.
The second, consisting of the same procedure without H2SO4 (12M) treatment, yielded
only hemicellulose and free monomers. Consequently, the cellulose content was calculated by
subtraction of the results obtained for the first experiment from those obtained for the second
Page 4 of 18
A mixture of ten monosaccharides (ribose, arabinose, xylose, rhamnose, fucose, glucose,
mannose and galactose, lyxose and allose) was used as external standard for compound
identification through peak retention times and for individual response coefficient
determination. Analyses gave an analytical precision between 10 to 15 % .
2.4. Carbohydrate analysis by preparative thermochemolysis
Each monosaccharide standard and cellulose (10 mg) was placed in a ceramic boat after
one hour moistening with 2 ml of a 50% (w/w) aqueous solution of tetramethylammonium
hydroxide (TMAH). Each sample was transferred in a Pyrex® tube (70 cm length, 3 cm i.d.)
and heated at 400°C (30 min isothermal period) in a tubular furnace. Thermochemolysis
products were swept by nitrogen (flow rate: 100 ml.min-1) to a trap containing
dichloromethane. After partial evaporation of the solvent under reduce pressure, trapped
pyrolysates were analysed by GC-MS using a Trace GC Thermo Finnigan (split injector,
250°C; FID, 300°C) with a fused silica capillary column (Supelco Equity 5%, 30 m length,
0.25 mm i.d., 0.25 µm film thickness) and helium as carrier gas. The oven was initially kept at
60 °C for 1 min, next it was heated at a rate of 5 °C/min to 300 °C and maintained at that
temperature for 15 min. The column was coupled to a Finnigan Trace MS quadrupole mass
spectrometer (ionization energy 70 eV, mass range m/z 45–600, cycle time 1 s). Peak
integration for the permethylated deoxy aldonic acids was performed in the extracted ion
chromatogram at m/z 129 and at m/z 173 for those from cellulose. Thermochemolysis
conditions for the analysis of carbohydrates in peat samples (90 mg) are identical as above.
The various products were identified on the basis of their GC retention times, their mass
spectra (comparison with standards) and literature data. Semi-quantification was achieved by
comparison of the peak area of a chosen permethylated isosaccharinic acid product specific to
a type of carbohydrate with the peak area of the same permethylated isosaccharinic acids
obtained after TMAH thermochemolysis of model compounds. Fig. 3 shows the extracted ion
chromatograms at m/z 129 for an aldohexose (glucose), an aldopentose (xylose) and a 6-
deoxyhexoses (fucose). With the same approach, the abundance of cellulose was estimated
following the mass fragment m/z 173 response for each peat sample.
Page 5 of 18
Fig. 4 presents the depth distributions of carbohydrate concentrations within the peat core released both by hydrolysis with H2SO4 and TMAH thermochemolysis.
3.1 H2SO4 hydrolysis
173 The highest concentration of galactose is in the upper part of the acrotelm (47 mg.g-1). Its
concentration shows a distinct decrease in the water table zone (around 20 cm) (37 mg.g-1)
before the concentrations increase to 46 mg.g-1 at 30 cm before decreasing again to 22 mg.g-1
in the remaining part of the core. Gulose was not detected.
As for galactose, the concentration of glucose, mannose and allose are higher in the acro-
and mesotelm and lower in the catotelm. In the uppermost subsurface layer, concentration
was 74 mg.g-1 and increased to 88 mg.g-1 at 15 cm, then regularly decreased to around 55
mg.g-1 at 50 cm.
The concentration of fucose shows a slight increase between the subsurface and the upper
part of the mesotelm with values around ca. 4.2 mg.g-1, before the concentration decreases to
around 2 mg.g-1 at the upper part of the catotelm. Finally an increase to 5 mg.g-1 was observed
in the deepest part of the core. The concentration of rhamnose shows relatively constant
values of 12–14 mg.g-1 in the acrotelm and mesotelm before decreasing to 7 mg.g-1 in the
The aldopentoses (mainly xylose and arabinose) content slightly increases from the
subsurface (55 mg.g-1) to the end of the acrotelm (70 mg.g-1) before decreasing in the
mesotelm (54 mg.g-1) and then decreasing with depth (from 72 to 37 mg.g-1) in the lower
Cellulose concentration increases through the acrotelm (from 124 to 161 mg.g-1) before
strongly decreasing in the uppermost mesotelm to 66 mg.g-1 and dropping to relatively
constant values around 105 mg.g-1 in the catotelm.
3.2 TMAH Thermochemolysis
After TMAH thermochemolysis, series of permethylated deoxy aldonic acids were
identified in the six peat samples. In all samples, the main permethylated deoxy aldonic acids
observed arose from cellulose and from free forms of glucose (and mannose/allose). Free
forms of aldopentoses, 6-deoxyhexoses and galactose (with gulose and idose) were detected
in lower amounts (Fig. 4).
Page 6 of 18
In the acrotelm (0 to 20 cm), all the permethylated deoxy aldonic acids products of free
carbohydrates decrease with depth (from 12.0 to 6.7 mg.g-1 for aldopentoses, from 22.8 to
11.4 mg.g-1 for glucose, mannose and allose, from 4.4 to 1.8 mg.g-1 for galactose, from 1.0 to
0.7 mg.g-1 for rhamnose and from 0.9 to 0.5 mg.g-1 for fucose). Cellulose concentration
decreases also with depth from 546 to 211 mg.g-1. Through the mesotelm, the concentration of aldopentoses, rhamnose and fucose continues
to decrease to values around 5.0, 0.4 and 0.3 mg.g-1 respectively. Glucose (with mannose and
allose), galactose and cellulose concentrations drop in the uppermost mesotelm and decrease
again (from 20.8 to 9.5, from 3.2 to 2.2 and from 396 to 312 mg.g-1 respectively).
All the concentrations of free carbohydrates show a distinct increase in the deepest part of
the peat core (the catotelm) to values higher than in the upper part of the acrotelm. The
concentration of cellulose increases in lower proportion up to ca. 447 mg.g-1 (Fig. 4).
215 4. Discussion
Acid hydrolysis carbohydrates showed high amounts in agreement with previous works
. Amounts of carbohydrates, and especially of hemicellulose sugars content, are almost
constant in the first 20 cm denoting a high preservation of these biopolymers (Fig. 4). With
depth, the substantial variations in cellulose sugars, that are the structural sugars of plant
tissues, reflect that this polymer is a prime target of degradation.
Carbohydrates monomers could also be used to infer vegetation communities changes. As
an example, some sugars such as galactose and rhamnose are considered as mosses indicators
[22, 25] whereas arabinose and xylose (aldopentoses) are considered as sedges indicators [8,
21-24]. Therefore, carbohydrates patterns with depth tended to indicate a decrease of mosses
contribution and therefore, an enhanced sedges contribution to organic matter inputs with
Similar distribution was not apparent for the TMAH results. With thermochemolysis,
amounts of all the analysed free carbohydrate types and cellulose decrease with depth in the
acrotelm before increasing in the catotelm. Poor agreement between the two methods occurs
because of inherent differences in chemolytic mechanisms, which resulted in bias in detection
of carbohydrates pools. Specifically, whereas the acid hydrolysis method is a classical way
for the analysis of almost all types of carbohydrates, the TMAH thermochemolysis allows the
specific analyses of free (or terminal) carbohydrates. As a consequence, amounts of non-
Page 7 of 18
cellulosic carbohydrates obtained by TMAH thermochemolysis are lower than those obtained
by the acid hydrolysis method. Contrary to acid hydrolysis, TMAH thermochemolysis is able to cleave common ester and
ether bonds and to methylate acidic functional groups. Hydrolysable ester and labile ether
bonds are present in acid-insoluble substances such as biopolymers (i.e. lignin, cutins, waxes
or tannins) and products formed during decomposition (i.e. humic substances) [28-29].
Because of the capability of TMAH thermochemolysis to cleave macromolecular structures
(i.e. cleavage of -O-4 bonds in lignin [30-32]), it allows the analyses of compounds
entrapped in macromolecular network . It results in the possible recovery of entrapped
carbohydrates and consequently in a greater cellulosic carbohydrates yield than for acid
In the oxic acrotelm, free carbohydrates and cellulose decreased. This might be link with
decomposition dynamic occurring in the oxic acrotelm. With depth, organic matter was more
decomposed. Therefore, deeper peat layers present lower yields of decomposable organic
matter than in the upper part where fresh plant inputs occurred. In the oxic acrotelm, TMAH
thermochemolysis of carbohydrates might thus reflected a decrease of decomposability
potential of organic matter with depth. Below the oxic acrotelm, the mesotelm is considered
as the compartment where water-level changes  and were peat decomposition might be
enhanced . At this depth, cellulose, glucose and galactose presented enhanced amounts at
ca. 20 cm depth. Therefore, TMAH thermochemolysis of carbohydrates might reflect this
enhanced decomposition dynamic of the mesotelm by the way of an increased of available
free carbohydrates and cellulose amounts under microbial activity. Finally in the lower anoxic
part, i.e. the catotelm, simple carbohydrates and cellulose amounts increased. The catotelm is
considered as the deeper peat compartment characterised by low decomposition processes and
by the accumulation of refractory compounds. Among these refractory biopolymers, lignin
forms a resistant shield around cellulose to form lignocellulose in plant cell walls [35-36]. As
also suggested by the acid analysis of sugars, the increase with depth of cellulose analysed by
TMAH thermochemolysis could indicate therefore a greater contribution from vascular plants
(i.e. Eriophorum spp.) with increasing age. Peatland evolution involves a number of dynamic
stages characterised by specific plant communities, changing from a fen characterised by the
predominance of Cyperaceae spp. to a raised bog with vegetation dominated by Sphagnum
spp. communities . Our results could thus document this typical change in peatland
evolution (from sedges in the bottom to Sphagnum spp. in the top).
Page 8 of 18
On the other hand, a micro-morphological characterisation of the same peat core  has
shown that the relative abundance of well-preserved tissues decreased with increasing depth
(from 66 to 11%) while mucilage contents (partly derived from in situ microbial syntheses 
increased (from 7 to 30%). In addition, the relative amounts of the acid-insoluble organic
matters increase gradually as the decomposition proceeds . Therefore, the increased
amounts of refractory neosynthetic organic compounds (the so-called humic substances) with
depth could be another way to explain the enhanced amount of carbohydrates obtained by
TMAH thermochemolysis in the catotelm. In such a case, refractory neosynthetic organic
compounds might act as a trap for carbohydrates. Because of the lack of complementary
information, we can not argue if the enhanced carbohydrates amounts were due to past
vegetation changes and/or to past humification processes. Combined with other analyses,
TMAH thermochemolysis of carbohydrates might be considered as a useful tool to provide
information about recent organic matter decomposition but also on past depositional
283 5. Conclusion
When compared with the widely used acid hydrolysis developed for carbohydrates
analysis, the application of TMAH thermochemolysis to our sediment samples did not
analyse hemicellulosic carbohydrates and discriminate each individual carbohydrates.
However, TMAH thermochemolysis allows the analysis of a cellulose pool hidden to acid
hydrolysis and the specific analysis of free and terminal carbohydrates. As a consequence,
we caution against making simple direct comparisons of thermochemolysis data with data
generated from acid hydrolysis because of the different mechanisms involved in each
process. Nevertheless, and because of their differences, they would be viewed and used as
pertinent complementary methods for the analysis of carbohydrates in complex
 W. Amelung, M.V. Cheshire, G. Guggenberger, Determination of neutral and acidic
sugars in soil by capillary gas liquid chromatography after trifluoroacetic acid hydrolysis, Soil
Biol. Biochem. 28 (1996) 1631-1639.
Page 9 of 18
 J.E. Modzeleski, W.A. Laurie, B. Nagy, Carbohydrates from Santa Barbara Basin
sediments: gas chromatographic mass spectrometric analysis of trimethylsilyl derivatives,
Geochim. Cosmochim. Acta 35 (1971) 825-838.
 G.L. Cowie, J.I. Hedges, Carbohydrate sources in a coastal marine environment, Geochim.
Cosmochim. Acta 48 (1984) 2075-2087.
 S. Hu, D.C. Coleman, M.H. Beare, P.F. Hendrix, Soil carbohydrates in aggrading and
degrading agroecosystems: influences of fungi and aggregates, Agric. Ecosyst. Environ. 54
 N. Koivula, K. Hänninen, Concentrations of monosaccharides in humic substances in the
early stages of humification, Chemosphere 44 (2001) 271-279.
 P. Rovira, V.R. Vallejo, Labile, recalcitrant, and inert organic matter in Mediterranean
forest soils, Soil Biol. Biochem. 39 (2007) 202-215.
 S. Ogier, J.-R. Disnar, P. Albéric, G. Bourdier, Neutral carbohydrate geochemistry of
particulate material (trap and core sediments) in an eutrophic lake (Aydat, France), Org.
Geochem. 32 (2001) 151-162.
 S. Bourdon, F. Laggoun-Défarge, J.-R. Disnar, O. Maman, B. Guillet, S. Derenne, C.
Largeau, Organic matter sources and early diagenetic degradation in a tropical peaty marsh
(Tritivakely, Madagascar), Org. Geochem. 31 (2000) 421-438.
 C. Rumpel, M.-F. Dignac, Chromatographic analysis of monosaccharides in a forest soil
profile: Analysis by gas chromatography after trifluoroacetic acid hydrolysis and reduction-
acetylation, Soil Biol. Biochem. 38 (2006) 1478-1481.
 S. Spielvogel, J. Prietzel, I. Kögel-Knabner, Changes of lignin phenol and neutral sugar
pools in different soil types of a high-elevation forest ecosystem 25 years after forest dieback
Soil Biol. Biochem. 39 (2007) 655-668.
 F. Shadkami, R. Helleur, Recent applications in analytical thermochemolysis, J. Anal.
Appl. Pyrol. 89 (2010) 2-16.
 L. Grasset, A. Amblès, Structural study of soil humic acids and humin using a new
preparative thermochemolysis technique, J. Anal. Appl. Pyrol. 47 (1998) 1-12.
 D. Fabbri, R. Helleur, Characterisation of the tetramethylammonium hydroxide
thermochemolysis products of carbohydrates, J. Anal. Appl. Pyrol. 49 (1999) 277-293.
 L. Grasset, P. Rovira, A. Amblès, TMAH-preparative thermochemolysis for the
characterization of organic matter in densimetric fractions of a Mediterranean forest soil, J.
Anal. Appl. Pyrol. 1-2 (2009) 435-441.
Page 10 of 18
 D.J. Clifford, D.M. Carson, D.E. McKinney, M. Bortiatynski, P.G. Hatcher, A new rapid
technique for the characterization of lignin in vascular plants: thermochemolysis with
tetramethylammonium hydroxide (TMAH), Org. Geochem. 23 (1995) 169-175.
 A. Gauthier, S. Derenne, C. Largeau, L. Dupont, E. Guillon, J. Dumonceau, M.
Aplincourt, Comparative study of ligno-cellulosic material from wheat straw and of pure and
mixed standard compounds via solid state 13C NMR spectroscopy, conventional pyrolysis and
TMAH thermochemolysis, J. Anal. Appl. Pyrol. 67 (2003) 277-293.
 B. Chefetz, Y. Chen, C.E. Clapp, P.G. Hatcher, Characterization of organic matter in
soils by thermochemolysis using tetramethylammonium hydroxide (TMAH), Soil Sci. Soc.
Am. J. 64 (2000) 583-589.
 C. Schwarzinger, On the mechanism of thermally assisted hydrolysis and methylation of
carbohydrates: the contribution of aldol and retroaldol reactions, J. Anal. Appl. Pyrol. 68-69
 I. Tanczos, C. Schwarzinger, H. Schmidt, J. Balla, THM-GC/MS analysis of model
uronic acids of pectin and hemicellulose, J. Anal. Appl. Pyrol. 68-69 (2003) 151-162.
 J. Bleton, P. Mejanelle, J. Sansoulet, S. Goursaud, A. Tchapla, Characterization of
neutral sugars and uronic acids after methanolysis and trimethyl-silylation for recognition of
plant gums, J. Chromatogr. A 720 (1996) 27-49.
 R.J. Wicks, M.A. Moran, L.J. Pittman, R.E. Hodson, Carbohydrate signatures of aquatic
macrophytes and their dissolved degradation products as determined by a sensitive high-
performance ion chromatography method, Appl. Environ. Microbiol. 57 (1991) 3135-3143.
 L. Comont, F. Laggoun-Défarge, J.-R. Disnar, Evolution of organic matter indicators in
response to major environmental changes: the case of a formerly cutover peatbog (Le Russey,
Jura Mountains, France), Org. Geochem. 37 (2006) 1736-1751.
 M.E.C. Moers, J.J. Boon, J.W. De Leeuw, M. Baas, P.A. Schenck, Carbohydrate
speciation and Py-MS mapping of peat samples from a subtropical open marsh environment,
Geochim. Cosmochim. Acta 53 (1989) 2011-2021.
 M.E.C. Moers, M. Baas, J.W. De Leeuw, J.J. Boon, P.A. Schenck, Occurrence and origin
of carbohydrates in peat samples from a red mangrove environment as reflected by
abundances of neutral monosaccharides, Geochim. Cosmochim. Acta 54 (1990) 2463-2472.
 Z.A. Popper and S.C. Fry, Primary cell wall composition of bryophytes and charophytes,
Ann. Bot. 91 (2003) 1-12.
 V.E.J. Jassey, G. Chiapusio, D. Gilbert, A. Buttler, M.-L. Toussaint, P. Binet.,
Experimental climate effect on seasonal variability of polyphenol/phenoloxidase interplay
Page 11 of 18
along a narrow fen-bog gradient in Sphagnum fallax, Global Change Biology (2011) DOI
 P.O. Bethge, C. Holmström, S. Juhlin, Quantitative gas chromatography of mixtures of
simple sugars. Svensk Papperst. 69 (1966) 60-63.
 W. Zech, M.-B. Johansson, L. Haumaier, R.L. Malcolm, CPMAS
spectra of spruce and pine litter and of the Klason lignin fraction at different stages of
decomposition, Z. Pflanzenern. Bodenk. 150 (1987) 262-265.
 C.M. Preston, J.R. Nault, J.A. Trofymow, Chemical Changes During 6 Years of
Decomposition of 11 Litters in Some Canadian Forest Sites. Part 2.
State 13C NMR Spectroscopy and the Meaning of ―Lignin‖, Ecosystems 12 (2009) 1078-1102.
chromatography/mass spectrometry, J. Anal. Appl. Pyrol 35 (1995) 93-107.
 P.G. Hatcher, M.A. Nanny, R.D. Minard, S.D. Dible, D.M. Carson, Comparison of two
thermochemolytic methods for the analysis of lignin in decomposing gymnosperm wood: the
CuO oxidation method and the method of thermochemolysis with tetramethylammonium
hydroxide (TMAH), Org. Geochem. 23 (1995), 881-888.
 L.A. Wysocki, T.R. Filley, T.S. Bianchi, Comparison of two methods for the analysis of
lignin in marine sediments: CuO oxidation versus tetramethylammonium hydroxide (TMAH)
thermochemolysis, Org. Geochem. 39 (2008) 1454-1461.
 R.S. Clymo, C.L. Bryant, Diffusion and mass flow of dissolved carbon dioxide, methane,
and dissolved organic carbon in a 7-m deep raised peat bog, Geochim. Cosmochim. Acta 72
 A. Haraguchi, C. Hasegawa, A. Hirayama, H. Kojima, Decomposition activity of peat
soils in geogenous mires in Sasakami, central Japan, Eur. J. Soil Biol. 39 (2003) 191-196.
 T. Osono, Ecology of ligninolytic fungi associated with leaf litter decomposition, Ecol.
Res. 22 (2007) 955-974.
 R.C. Cooke, A.D.M. Whipps, Ecophysiology of fungi, Blackwell, Oxford, 1993.
 O. Manneville, V. Vergne, O., Villepoux, Le monde des tourbières et des marais : France,
Suisse, Belgique et Luxembourg,. Delachaux et Niestlé, Paris-Lausanne, 1999.
 F. Delarue, F. Laggoun-Défarge, J.R. Disnar, N. Lottier, S. Gogo, Organic matter sources
and decay assessment in a Sphagnum-dominated peatland (Le Forbonnet, Jura Mountains,
France): impact of moisture conditions, Biogeochem. (2011) DOI 10.1007/s10533-010-9410-
C Abundance, Solid-
C NMR and IR
Page 12 of 18
 F. Laggoun-Défarge, S. Bourdon, C. Chenu, Etude des transformations morphologiques
précoces des tissues végétaux de tourbe. Apport du marquage histochimique en MET et du
cryo-MEB haute resolution, in: F. Elsass F, A.M. Jaunet (Eds.), Structure et ultrastructure des
sols et des organismes vivants, INRA, Paris, 1999, pp. 169-182.
 C. McClaugherty, B. Berg, Cellulose, lignin and nitrogen concentration as rate regulating
factors in late stages of forest litter decomposition, Pedobiologia 30 (1987) 101-112.
Page 13 of 18
Fig. 1: Mechanism proposed by Fabbri and Helleur (1999)  of the formation of methylated saccharinic acids from the TMAH thermochemolysis of glucose.
Fig. 2: Peat core and depths of sampling.
Fig. 3: Extracted Ion Chromatograms at m/z 129 of permethylated saccharinic acids
obtained after TMAH thermochemolysis of glucose (a), xylose (b) and fucose (c) and at m/z
173 of permethylated saccharinic acids obtained after TMAH thermochemolysis of cellulose
(d). Peaks in gray are peaks corresponding to permethylated saccharinic acids used for
Fig. 4 : Depth distributions of carbohydrate concentrations within the peat core released
after H2SO4 hydrolysis (up) and TMAH thermochemolysis (down) (in mg/g of dry sample).
Page 14 of 18
OMe HO OH HO
Base - H2O
Page 15 of 18
20.0-22.5 cm 25.0-27.5 cm 40.0-42.5 cm
Page 16 of 18
Ac Time (min)
b) Time (min)
Page 17 of 18
Glucose Mannose, Allose
Page 18 of 18