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�
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Manuscript
Tetramethylammonium hydroxide thermochemolysis for the analysis of cellulose and free carbohydrates in a peatbog
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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] Abstract
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We have compared TMAH thermochemolysis with the classical method using acid hydrolysis
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for carbohydrates analysis in a peat core. Even if TMAH thermochemolysis does not analyse
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hemicellulosic carbohydrates and discriminate each individual carbohydrate sensu stricto, it
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allows the analysis of a cellulose pool hidden to acid hydrolysis and the specific analysis of
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free and terminal carbohydrates. Simple direct comparisons of thermochemolysis data with
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data generated from acid hydrolysis cannot be done because of the different mechanisms
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involved in each process. TMAH thermochemolysis must be viewed and used as a pertinent
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and complementary method for the analysis of carbohydrates protected and trapped by the
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organic matter in complex environmental systems.
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24 1. Introduction
Because of their ubiquity and abundance, carbohydrates are potentially useful compounds
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in elucidating sources, processes and pathways of biologically important organic materials in
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natural environments.
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Acid hydrolysis is currently used to liberate carbohydrates from soils. It involves acid
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hydrolysis and purification before their analysis by liquid chromatography or by gas
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chromatography after derivatization. Recommended procedures either use H2SO4 [1-6], HCl
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[1, 6-8] or TFA [1, 9-10] to cleave glycosidic bonds of polysaccharides yielding sugar
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monomers. These methods can be applied to a wide range of soil samples and are easy to
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perform but yet time-consuming.
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Tetramethylammonium hydroxide (TMAH) is the most common reagent used for
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thermochemolysis (more than 90% of published thermochemolysis applications have used
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TMAH [11]). It is used for the analysis of complex and intractable samples such as soils and
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sediments. For that kind of samples, TMAH thermochemolysis provides useful information
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simultaneously on a wide range of compounds related to lipids, lignins, tannins, proteins and
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carbohydrates markers. Due to its capability to cleave common ester and ether bonds and to
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methylate acidic functional groups, TMAH thermochemolysis allows also the analyses of
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compounds entrapped in macromolecular network [12]. However, few studies have identified these products in soil samples [13-14]. It could be
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partly due to the relative poor sensitivity of TMAH thermochemolysis to carbohydrates both
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at high temperature (up to 600°C) [15-16] or with extended reaction time (250°C during 30
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min in sealed tube) [17].
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On the other hand, several studies have shown that TMAH thermochemolysis of
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individual sugars releases, beside products formed by recombination of previously cleaved
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fragments, 3-deoxyaldonic acids methyl esters resulting from the isomerisation of the C-2
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position and dehydration of the C-3 position. These products have conserved their original
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conformation at the C-4 and C-5 positions [13, 18-19]. As a consequence, aldohexoses such
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as glucose, mannose and allose give identical 3-deoxyaldonic acids methyl esters but different
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ones from their C-4 epimers (i.e. galactose, gulose and idose). In the same way, the 6-
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deoxyhexoses, fucose and rhamnose (the C-4 epimer of fucose), give different saccharinic
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acids methyl esters. For the same reason, aldopentoses (xylose, arabinose, ribose and lyxose)
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give the same saccharinic acid methyl esters (Fig. 1). The methylated forms of these
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saccharinic products have mass spectra with m/z 129 as base peak (for the interpretation of the
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electron impact MS fragmentation of permethylated saccharinic acids see Fabbri and Helleur
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(1999) [13] and Bleton et al. (1996) [20]). Furthermore, although their mass spectra present
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no evident differences, they present different chromatographic behaviour with different
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retention times in gas chromatography. Moreover, TMAH thermochemolysis of cellulose
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produces a specific epimeric pair of methylated isosaccharinic acids producing specific ions
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in EI mass spectroscopy (i.e. m/z 173) [13]. Then, to detect monosaccharides and glycosidic
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units from cellulose with TMAH thermochemolysis, single ion monitoring at m/z 129 and 173
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can be used to reveal their presence in complex materials.
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Soil organic matter is composed of more or less altered and inherited biochemical
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compounds
such as
hemicellulose, cellulose and microbial
sugars (i.e. mainly
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exopolysaccharides). Depending on organic matter sources and degradation, carbohydrate
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monomers exhibit distinctive composition patterns. Ombrotrophic peat bogs are covered
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mainly by Sphagnum spp. with Eriophorum as the dominant vascular species. Cyperaceae are
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rich in xylose (one of the most abundant aldopentoses in living kingdom) [8, 21] as well as in
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arabinose [22-24]. Conversely, galactose, mannose and rhamnose are adequate indicators for
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mosses, in particular Sphagnum spp. [22, 25]. In addition, fucose could be considered as a
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microbial marker in peat bogs [22]. At 400°C, a recent study has shown that the identification of carbohydrates derivatives in
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soil samples is possible with TMAH thermochemolysis [14]. In addition, TMAH
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thermochemolysis can be applied to precise and large quantities of material (up to 1g) since
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an off-line preparative technique was developed [12].
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Samples collected at different depths of a peat core were submitted to TMAH
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thermochemolysis.
Under the same thermochemolysis conditions, cellulose and the most abundant
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monosaccharides encountered in peat bog were submitted to thermochemolysis allowing their
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assignment among the peat samples products based both on the mass spectra and on the
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retention times in gas chromatography of their thermochemolysis derivatives. Despite
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unavoidable matrix effects, this off-line preparative thermochemolysis also allows a semi-
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quantification of thermochemolysis products when known quantities of standards were
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analysed in the same conditions than the studied complex samples.
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Results were compared with those obtained using a classical method for carbohydrates
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analyses (acid hydrolysis with H2SO4) to have insight on the capability of TMAH
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thermochemolysis for the carbohydrate analyses in soils and sediments.
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2. Materials and methods
2.1. Chemicals
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Aldopentoses (D-xylose, L-arabinose, D-ribose), aldohexoses (D-glucose, D-mannose, D-
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galactose), deoxyhexoses (L-rhamnose, L-fucose), cellulose, deoxy-6-glucose, H2SO4, CaCO3
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CH3OH, CH2Cl2, LiClO4 and TMAH were purchased from Sigma-Aldrich (St Louis, USA)
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and BSTFA+TMCS (99:1) from Supelco (Bellefonte, USA).
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2.2. Peat samples
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Samples were collected in June 2008 at the open bog part of an undisturbed Sphagnum-
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dominated mire in the Jura Mountains (Le Forbonnet peatland, France), which has been
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described in detail [26]. The site is protected by the EU Habitat Directive of Natura 2000 and
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has been classified as a Region Natural Reserve for more than 20 yr. Annual precipitation is
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about 1300–1500 mm per year, with a mean annual air temperature of 7–8 °C. Samples were
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collected at different depths: (i) two samples were collected in the upper oxic part (acrotelm),
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(ii) two in the water table zone (mesotelm) and (iii) two in the anoxic part (catotelm) (Fig. 2).
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They were air dried (40°C), finely ground and stored at -20°C before further analysis.
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2.3. Carbohydrate analysis by hot water extraction and H2SO4 hydrolysis
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The detailed H2SO4 hydrolysis method has been described elsewhere [22]. The analysis is
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carried out in two experiments. The first operating procedure for total sugar analysis can be
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summarized as follows: 1 ml of H2SO4 (12M) was added to 20 to 30 mg of dry peat in a
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Pyrex® test tube. After 16 h at room temperature, the samples were diluted with 9 ml of H2O
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to a 1.2 M concentration in H2SO4. The tube was tightly closed under vacuum and heated at
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100°C for 4 h. After cooling, deoxy-6-glucose (0.4 mg.ml-1 in water) was added as internal
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standard [21]. The sample was subsequently neutralised with CaCO3. The precipitate was
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removed by centrifugation and the supernatant evaporated to dryness. The sugars were then
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dissolved in CH3OH and the solution purified by centrifugation. After transferring the
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solution to another vessel, the solvent was evaporated under vacuum. The sugars were
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dissolved in pyridine containing 1 % (v/v) of LiClO4 and left 16 h at 60°C for anomer
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equilibration [27]. In both cases, they were silylated by BSTFA+TMCS (99:1) and analysed
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using a Perkin–Elmer AutoSystem XL GC (split injector, 240°C; flame ionization detector
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(FID), 300°C) with a fused silica capillary column (CPSil5CB, 25 m length, 0.25 mm i.d.,
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0.25 µm film thickness) and helium as carrier gas. The GC was temperature programmed
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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
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finally at 20°C.min-1 to 310°C and maintained at that temperature for 10 min.
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The second, consisting of the same procedure without H2SO4 (12M) treatment, yielded
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only hemicellulose and free monomers. Consequently, the cellulose content was calculated by
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subtraction of the results obtained for the first experiment from those obtained for the second
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one.
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A mixture of ten monosaccharides (ribose, arabinose, xylose, rhamnose, fucose, glucose,
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mannose and galactose, lyxose and allose) was used as external standard for compound
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identification through peak retention times and for individual response coefficient
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determination. Analyses gave an analytical precision between 10 to 15 % [22].
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2.4. Carbohydrate analysis by preparative thermochemolysis
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Each monosaccharide standard and cellulose (10 mg) was placed in a ceramic boat after
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one hour moistening with 2 ml of a 50% (w/w) aqueous solution of tetramethylammonium
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hydroxide (TMAH). Each sample was transferred in a Pyrex® tube (70 cm length, 3 cm i.d.)
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and heated at 400°C (30 min isothermal period) in a tubular furnace. Thermochemolysis
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products were swept by nitrogen (flow rate: 100 ml.min-1) to a trap containing
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dichloromethane. After partial evaporation of the solvent under reduce pressure, trapped
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pyrolysates were analysed by GC-MS using a Trace GC Thermo Finnigan (split injector,
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250°C; FID, 300°C) with a fused silica capillary column (Supelco Equity 5%, 30 m length,
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0.25 mm i.d., 0.25 µm film thickness) and helium as carrier gas. The oven was initially kept at
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60 °C for 1 min, next it was heated at a rate of 5 °C/min to 300 °C and maintained at that
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temperature for 15 min. The column was coupled to a Finnigan Trace MS quadrupole mass
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spectrometer (ionization energy 70 eV, mass range m/z 45–600, cycle time 1 s). Peak
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integration for the permethylated deoxy aldonic acids was performed in the extracted ion
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chromatogram at m/z 129 and at m/z 173 for those from cellulose. Thermochemolysis
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conditions for the analysis of carbohydrates in peat samples (90 mg) are identical as above.
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The various products were identified on the basis of their GC retention times, their mass
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spectra (comparison with standards) and literature data. Semi-quantification was achieved by
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comparison of the peak area of a chosen permethylated isosaccharinic acid product specific to
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a type of carbohydrate with the peak area of the same permethylated isosaccharinic acids
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obtained after TMAH thermochemolysis of model compounds. Fig. 3 shows the extracted ion
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chromatograms at m/z 129 for an aldohexose (glucose), an aldopentose (xylose) and a 6-
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deoxyhexoses (fucose). With the same approach, the abundance of cellulose was estimated
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following the mass fragment m/z 173 response for each peat sample.
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3. Results
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Fig. 4 presents the depth distributions of carbohydrate concentrations within the peat core released both by hydrolysis with H2SO4 and TMAH thermochemolysis.
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3.1 H2SO4 hydrolysis
173 The highest concentration of galactose is in the upper part of the acrotelm (47 mg.g-1). Its
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concentration shows a distinct decrease in the water table zone (around 20 cm) (37 mg.g-1)
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before the concentrations increase to 46 mg.g-1 at 30 cm before decreasing again to 22 mg.g-1
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in the remaining part of the core. Gulose was not detected.
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As for galactose, the concentration of glucose, mannose and allose are higher in the acro-
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and mesotelm and lower in the catotelm. In the uppermost subsurface layer, concentration
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was 74 mg.g-1 and increased to 88 mg.g-1 at 15 cm, then regularly decreased to around 55
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mg.g-1 at 50 cm.
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The concentration of fucose shows a slight increase between the subsurface and the upper
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part of the mesotelm with values around ca. 4.2 mg.g-1, before the concentration decreases to
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around 2 mg.g-1 at the upper part of the catotelm. Finally an increase to 5 mg.g-1 was observed
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in the deepest part of the core. The concentration of rhamnose shows relatively constant
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values of 12–14 mg.g-1 in the acrotelm and mesotelm before decreasing to 7 mg.g-1 in the
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catotelm.
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The aldopentoses (mainly xylose and arabinose) content slightly increases from the
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subsurface (55 mg.g-1) to the end of the acrotelm (70 mg.g-1) before decreasing in the
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mesotelm (54 mg.g-1) and then decreasing with depth (from 72 to 37 mg.g-1) in the lower
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anoxic part.
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Cellulose concentration increases through the acrotelm (from 124 to 161 mg.g-1) before
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strongly decreasing in the uppermost mesotelm to 66 mg.g-1 and dropping to relatively
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constant values around 105 mg.g-1 in the catotelm.
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3.2 TMAH Thermochemolysis
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After TMAH thermochemolysis, series of permethylated deoxy aldonic acids were
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identified in the six peat samples. In all samples, the main permethylated deoxy aldonic acids
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observed arose from cellulose and from free forms of glucose (and mannose/allose). Free
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forms of aldopentoses, 6-deoxyhexoses and galactose (with gulose and idose) were detected
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in lower amounts (Fig. 4).
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In the acrotelm (0 to 20 cm), all the permethylated deoxy aldonic acids products of free
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carbohydrates decrease with depth (from 12.0 to 6.7 mg.g-1 for aldopentoses, from 22.8 to
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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
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0.7 mg.g-1 for rhamnose and from 0.9 to 0.5 mg.g-1 for fucose). Cellulose concentration
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decreases also with depth from 546 to 211 mg.g-1. Through the mesotelm, the concentration of aldopentoses, rhamnose and fucose continues
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to decrease to values around 5.0, 0.4 and 0.3 mg.g-1 respectively. Glucose (with mannose and
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allose), galactose and cellulose concentrations drop in the uppermost mesotelm and decrease
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again (from 20.8 to 9.5, from 3.2 to 2.2 and from 396 to 312 mg.g-1 respectively).
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All the concentrations of free carbohydrates show a distinct increase in the deepest part of
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the peat core (the catotelm) to values higher than in the upper part of the acrotelm. The
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concentration of cellulose increases in lower proportion up to ca. 447 mg.g-1 (Fig. 4).
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Acid hydrolysis carbohydrates showed high amounts in agreement with previous works
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[22]. Amounts of carbohydrates, and especially of hemicellulose sugars content, are almost
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constant in the first 20 cm denoting a high preservation of these biopolymers (Fig. 4). With
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depth, the substantial variations in cellulose sugars, that are the structural sugars of plant
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tissues, reflect that this polymer is a prime target of degradation.
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Carbohydrates monomers could also be used to infer vegetation communities changes. As
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an example, some sugars such as galactose and rhamnose are considered as mosses indicators
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[22, 25] whereas arabinose and xylose (aldopentoses) are considered as sedges indicators [8,
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21-24]. Therefore, carbohydrates patterns with depth tended to indicate a decrease of mosses
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contribution and therefore, an enhanced sedges contribution to organic matter inputs with
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increasing age.
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Similar distribution was not apparent for the TMAH results. With thermochemolysis,
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amounts of all the analysed free carbohydrate types and cellulose decrease with depth in the
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acrotelm before increasing in the catotelm. Poor agreement between the two methods occurs
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because of inherent differences in chemolytic mechanisms, which resulted in bias in detection
233
of carbohydrates pools. Specifically, whereas the acid hydrolysis method is a classical way
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for the analysis of almost all types of carbohydrates, the TMAH thermochemolysis allows the
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specific analyses of free (or terminal) carbohydrates. As a consequence, amounts of non-
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cellulosic carbohydrates obtained by TMAH thermochemolysis are lower than those obtained
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by the acid hydrolysis method. Contrary to acid hydrolysis, TMAH thermochemolysis is able to cleave common ester and
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ether bonds and to methylate acidic functional groups. Hydrolysable ester and labile ether
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bonds are present in acid-insoluble substances such as biopolymers (i.e. lignin, cutins, waxes
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or tannins) and products formed during decomposition (i.e. humic substances) [28-29].
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Because of the capability of TMAH thermochemolysis to cleave macromolecular structures
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(i.e. cleavage of -O-4 bonds in lignin [30-32]), it allows the analyses of compounds
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entrapped in macromolecular network [12]. It results in the possible recovery of entrapped
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carbohydrates and consequently in a greater cellulosic carbohydrates yield than for acid
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hydrolysis.
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In the oxic acrotelm, free carbohydrates and cellulose decreased. This might be link with
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decomposition dynamic occurring in the oxic acrotelm. With depth, organic matter was more
249
decomposed. Therefore, deeper peat layers present lower yields of decomposable organic
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matter than in the upper part where fresh plant inputs occurred. In the oxic acrotelm, TMAH
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thermochemolysis of carbohydrates might thus reflected a decrease of decomposability
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potential of organic matter with depth. Below the oxic acrotelm, the mesotelm is considered
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as the compartment where water-level changes [33] and were peat decomposition might be
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enhanced [34]. At this depth, cellulose, glucose and galactose presented enhanced amounts at
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ca. 20 cm depth. Therefore, TMAH thermochemolysis of carbohydrates might reflect this
256
enhanced decomposition dynamic of the mesotelm by the way of an increased of available
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free carbohydrates and cellulose amounts under microbial activity. Finally in the lower anoxic
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part, i.e. the catotelm, simple carbohydrates and cellulose amounts increased. The catotelm is
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considered as the deeper peat compartment characterised by low decomposition processes and
260
by the accumulation of refractory compounds. Among these refractory biopolymers, lignin
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forms a resistant shield around cellulose to form lignocellulose in plant cell walls [35-36]. As
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also suggested by the acid analysis of sugars, the increase with depth of cellulose analysed by
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TMAH thermochemolysis could indicate therefore a greater contribution from vascular plants
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(i.e. Eriophorum spp.) with increasing age. Peatland evolution involves a number of dynamic
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stages characterised by specific plant communities, changing from a fen characterised by the
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predominance of Cyperaceae spp. to a raised bog with vegetation dominated by Sphagnum
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spp. communities [37]. Our results could thus document this typical change in peatland
268
evolution (from sedges in the bottom to Sphagnum spp. in the top).
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On the other hand, a micro-morphological characterisation of the same peat core [38] has
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shown that the relative abundance of well-preserved tissues decreased with increasing depth
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(from 66 to 11%) while mucilage contents (partly derived from in situ microbial syntheses [39]
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increased (from 7 to 30%). In addition, the relative amounts of the acid-insoluble organic
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matters increase gradually as the decomposition proceeds [40]. Therefore, the increased
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amounts of refractory neosynthetic organic compounds (the so-called humic substances) with
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depth could be another way to explain the enhanced amount of carbohydrates obtained by
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TMAH thermochemolysis in the catotelm. In such a case, refractory neosynthetic organic
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compounds might act as a trap for carbohydrates. Because of the lack of complementary
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information, we can not argue if the enhanced carbohydrates amounts were due to past
279
vegetation changes and/or to past humification processes. Combined with other analyses,
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TMAH thermochemolysis of carbohydrates might be considered as a useful tool to provide
281
information about recent organic matter decomposition but also on past depositional
282
environments.
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283 5. Conclusion
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When compared with the widely used acid hydrolysis developed for carbohydrates
287
analysis, the application of TMAH thermochemolysis to our sediment samples did not
288
analyse hemicellulosic carbohydrates and discriminate each individual carbohydrates.
289
However, TMAH thermochemolysis allows the analysis of a cellulose pool hidden to acid
290
hydrolysis and the specific analysis of free and terminal carbohydrates. As a consequence,
291
we caution against making simple direct comparisons of thermochemolysis data with data
292
generated from acid hydrolysis because of the different mechanisms involved in each
293
process. Nevertheless, and because of their differences, they would be viewed and used as
294
pertinent complementary methods for the analysis of carbohydrates in complex
295
environmental systems.
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References
298 299
[1] W. Amelung, M.V. Cheshire, G. Guggenberger, Determination of neutral and acidic
300
sugars in soil by capillary gas liquid chromatography after trifluoroacetic acid hydrolysis, Soil
301
Biol. Biochem. 28 (1996) 1631-1639.
Page 9 of 18
[2] J.E. Modzeleski, W.A. Laurie, B. Nagy, Carbohydrates from Santa Barbara Basin
303
sediments: gas chromatographic mass spectrometric analysis of trimethylsilyl derivatives,
304
Geochim. Cosmochim. Acta 35 (1971) 825-838.
305
[3] G.L. Cowie, J.I. Hedges, Carbohydrate sources in a coastal marine environment, Geochim.
306
Cosmochim. Acta 48 (1984) 2075-2087.
307
[4] S. Hu, D.C. Coleman, M.H. Beare, P.F. Hendrix, Soil carbohydrates in aggrading and
308
degrading agroecosystems: influences of fungi and aggregates, Agric. Ecosyst. Environ. 54
309
(1995) 77-88.
310
[5] N. Koivula, K. Hänninen, Concentrations of monosaccharides in humic substances in the
311
early stages of humification, Chemosphere 44 (2001) 271-279.
312
[6] P. Rovira, V.R. Vallejo, Labile, recalcitrant, and inert organic matter in Mediterranean
313
forest soils, Soil Biol. Biochem. 39 (2007) 202-215.
314
[7] S. Ogier, J.-R. Disnar, P. Albéric, G. Bourdier, Neutral carbohydrate geochemistry of
315
particulate material (trap and core sediments) in an eutrophic lake (Aydat, France), Org.
316
Geochem. 32 (2001) 151-162.
317
[8] S. Bourdon, F. Laggoun-Défarge, J.-R. Disnar, O. Maman, B. Guillet, S. Derenne, C.
318
Largeau, Organic matter sources and early diagenetic degradation in a tropical peaty marsh
319
(Tritivakely, Madagascar), Org. Geochem. 31 (2000) 421-438.
320
[9] C. Rumpel, M.-F. Dignac, Chromatographic analysis of monosaccharides in a forest soil
321
profile: Analysis by gas chromatography after trifluoroacetic acid hydrolysis and reduction-
322
acetylation, Soil Biol. Biochem. 38 (2006) 1478-1481.
323
[10] S. Spielvogel, J. Prietzel, I. Kögel-Knabner, Changes of lignin phenol and neutral sugar
324
pools in different soil types of a high-elevation forest ecosystem 25 years after forest dieback
325
Soil Biol. Biochem. 39 (2007) 655-668.
326
[11] F. Shadkami, R. Helleur, Recent applications in analytical thermochemolysis, J. Anal.
327
Appl. Pyrol. 89 (2010) 2-16.
328
[12] L. Grasset, A. Amblès, Structural study of soil humic acids and humin using a new
329
preparative thermochemolysis technique, J. Anal. Appl. Pyrol. 47 (1998) 1-12.
330
[13] D. Fabbri, R. Helleur, Characterisation of the tetramethylammonium hydroxide
331
thermochemolysis products of carbohydrates, J. Anal. Appl. Pyrol. 49 (1999) 277-293.
332
[14] L. Grasset, P. Rovira, A. Amblès, TMAH-preparative thermochemolysis for the
333
characterization of organic matter in densimetric fractions of a Mediterranean forest soil, J.
334
Anal. Appl. Pyrol. 1-2 (2009) 435-441.
Ac
ce pt
ed
M
an
us
cr
ip t
302
Page 10 of 18
[15] D.J. Clifford, D.M. Carson, D.E. McKinney, M. Bortiatynski, P.G. Hatcher, A new rapid
336
technique for the characterization of lignin in vascular plants: thermochemolysis with
337
tetramethylammonium hydroxide (TMAH), Org. Geochem. 23 (1995) 169-175.
338
[16] A. Gauthier, S. Derenne, C. Largeau, L. Dupont, E. Guillon, J. Dumonceau, M.
339
Aplincourt, Comparative study of ligno-cellulosic material from wheat straw and of pure and
340
mixed standard compounds via solid state 13C NMR spectroscopy, conventional pyrolysis and
341
TMAH thermochemolysis, J. Anal. Appl. Pyrol. 67 (2003) 277-293.
342
[17] B. Chefetz, Y. Chen, C.E. Clapp, P.G. Hatcher, Characterization of organic matter in
343
soils by thermochemolysis using tetramethylammonium hydroxide (TMAH), Soil Sci. Soc.
344
Am. J. 64 (2000) 583-589.
345
[18] C. Schwarzinger, On the mechanism of thermally assisted hydrolysis and methylation of
346
carbohydrates: the contribution of aldol and retroaldol reactions, J. Anal. Appl. Pyrol. 68-69
347
(2003) 137-149.
348
[19] I. Tanczos, C. Schwarzinger, H. Schmidt, J. Balla, THM-GC/MS analysis of model
349
uronic acids of pectin and hemicellulose, J. Anal. Appl. Pyrol. 68-69 (2003) 151-162.
350
[20] J. Bleton, P. Mejanelle, J. Sansoulet, S. Goursaud, A. Tchapla, Characterization of
351
neutral sugars and uronic acids after methanolysis and trimethyl-silylation for recognition of
352
plant gums, J. Chromatogr. A 720 (1996) 27-49.
353
[21] R.J. Wicks, M.A. Moran, L.J. Pittman, R.E. Hodson, Carbohydrate signatures of aquatic
354
macrophytes and their dissolved degradation products as determined by a sensitive high-
355
performance ion chromatography method, Appl. Environ. Microbiol. 57 (1991) 3135-3143.
356
[22] L. Comont, F. Laggoun-Défarge, J.-R. Disnar, Evolution of organic matter indicators in
357
response to major environmental changes: the case of a formerly cutover peatbog (Le Russey,
358
Jura Mountains, France), Org. Geochem. 37 (2006) 1736-1751.
359
[23] M.E.C. Moers, J.J. Boon, J.W. De Leeuw, M. Baas, P.A. Schenck, Carbohydrate
360
speciation and Py-MS mapping of peat samples from a subtropical open marsh environment,
361
Geochim. Cosmochim. Acta 53 (1989) 2011-2021.
362
[24] M.E.C. Moers, M. Baas, J.W. De Leeuw, J.J. Boon, P.A. Schenck, Occurrence and origin
363
of carbohydrates in peat samples from a red mangrove environment as reflected by
364
abundances of neutral monosaccharides, Geochim. Cosmochim. Acta 54 (1990) 2463-2472.
365
[25] Z.A. Popper and S.C. Fry, Primary cell wall composition of bryophytes and charophytes,
366
Ann. Bot. 91 (2003) 1-12.
367
[26] V.E.J. Jassey, G. Chiapusio, D. Gilbert, A. Buttler, M.-L. Toussaint, P. Binet.,
368
Experimental climate effect on seasonal variability of polyphenol/phenoloxidase interplay
Ac
ce pt
ed
M
an
us
cr
ip t
335
Page 11 of 18
369
along a narrow fen-bog gradient in Sphagnum fallax, Global Change Biology (2011) DOI
370
10.1111/j1365-3486-2011-02437-x.
371
[27] P.O. Bethge, C. Holmström, S. Juhlin, Quantitative gas chromatography of mixtures of
372
simple sugars. Svensk Papperst. 69 (1966) 60-63.
373
[28] W. Zech, M.-B. Johansson, L. Haumaier, R.L. Malcolm, CPMAS
374
spectra of spruce and pine litter and of the Klason lignin fraction at different stages of
375
decomposition, Z. Pflanzenern. Bodenk. 150 (1987) 262-265.
376
[29] C.M. Preston, J.R. Nault, J.A. Trofymow, Chemical Changes During 6 Years of
377
Decomposition of 11 Litters in Some Canadian Forest Sites. Part 2.
378
State 13C NMR Spectroscopy and the Meaning of ―Lignin‖, Ecosystems 12 (2009) 1078-1102.
379
[30]
380
chromatography/mass spectrometry, J. Anal. Appl. Pyrol 35 (1995) 93-107.
381
[31] P.G. Hatcher, M.A. Nanny, R.D. Minard, S.D. Dible, D.M. Carson, Comparison of two
382
thermochemolytic methods for the analysis of lignin in decomposing gymnosperm wood: the
383
CuO oxidation method and the method of thermochemolysis with tetramethylammonium
384
hydroxide (TMAH), Org. Geochem. 23 (1995), 881-888.
385
[32] L.A. Wysocki, T.R. Filley, T.S. Bianchi, Comparison of two methods for the analysis of
386
lignin in marine sediments: CuO oxidation versus tetramethylammonium hydroxide (TMAH)
387
thermochemolysis, Org. Geochem. 39 (2008) 1454-1461.
388
[33] R.S. Clymo, C.L. Bryant, Diffusion and mass flow of dissolved carbon dioxide, methane,
389
and dissolved organic carbon in a 7-m deep raised peat bog, Geochim. Cosmochim. Acta 72
390
(2008) 2048-2066.
391
[34] A. Haraguchi, C. Hasegawa, A. Hirayama, H. Kojima, Decomposition activity of peat
392
soils in geogenous mires in Sasakami, central Japan, Eur. J. Soil Biol. 39 (2003) 191-196.
393
[35] T. Osono, Ecology of ligninolytic fungi associated with leaf litter decomposition, Ecol.
394
Res. 22 (2007) 955-974.
395
[36] R.C. Cooke, A.D.M. Whipps, Ecophysiology of fungi, Blackwell, Oxford, 1993.
396
[37] O. Manneville, V. Vergne, O., Villepoux, Le monde des tourbières et des marais : France,
397
Suisse, Belgique et Luxembourg,. Delachaux et Niestlé, Paris-Lausanne, 1999.
398
[38] F. Delarue, F. Laggoun-Défarge, J.R. Disnar, N. Lottier, S. Gogo, Organic matter sources
399
and decay assessment in a Sphagnum-dominated peatland (Le Forbonnet, Jura Mountains,
400
France): impact of moisture conditions, Biogeochem. (2011) DOI 10.1007/s10533-010-9410-
401
0
of
wood
by
ip t
cr
Characterisation
C Abundance, Solid-
pyrolysis
us
Challinor,
C NMR and IR
derivatisation—gas
Ac
ce pt
ed
M
an
J.M.
13
13
Page 12 of 18
402
[39] F. Laggoun-Défarge, S. Bourdon, C. Chenu, Etude des transformations morphologiques
403
précoces des tissues végétaux de tourbe. Apport du marquage histochimique en MET et du
404
cryo-MEB haute resolution, in: F. Elsass F, A.M. Jaunet (Eds.), Structure et ultrastructure des
405
sols et des organismes vivants, INRA, Paris, 1999, pp. 169-182.
406
[40] C. McClaugherty, B. Berg, Cellulose, lignin and nitrogen concentration as rate regulating
407
factors in late stages of forest litter decomposition, Pedobiologia 30 (1987) 101-112.
Ac
ce pt
ed
M
an
us
cr
ip t
408
Page 13 of 18
Figures Captions
Fig. 1: Mechanism proposed by Fabbri and Helleur (1999) [13] of the formation of methylated saccharinic acids from the TMAH thermochemolysis of glucose.
Fig. 2: Peat core and depths of sampling.
ip t
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
cr
173 of permethylated saccharinic acids obtained after TMAH thermochemolysis of cellulose
quantification.
us
(d). Peaks in gray are peaks corresponding to permethylated saccharinic acids used for
an
Fig. 4 : Depth distributions of carbohydrate concentrations within the peat core released
Ac
ce pt
ed
M
after H2SO4 hydrolysis (up) and TMAH thermochemolysis (down) (in mg/g of dry sample).
Page 14 of 18
Figure 1
O
O
O
OMe HO OH HO
Base - H2O
O
MeO OH-
HO
OH
TMAH
MeO
OH OH
OMe
Ac
ce pt
ed
M
an
us
cr
ip t
OH
OMe
Page 15 of 18
Figure 2
20.0-22.5 cm 25.0-27.5 cm 40.0-42.5 cm
Mesotelm
Catotelm
Ac
ce pt
ed
M
an
us
47.5-50.0 cm
ip t
12.5-15.0 cm
Acrotelm
cr
2.5-5.0 cm
Page 16 of 18
Relative Abundance
ed
Relative Abundance
ce pt
Ac Time (min)
us
an
c)
M
cr
Relative Abundance
b) Time (min)
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Relative Abundance
Figure 3
a)
d)
Time (min)
Time (min)
Page 17 of 18
Figure 4
Aldopentoses
Rhamnose
Fucose
Glucose Mannose, Allose
Galactose
Ac
ce pt
ed
M
an
us
cr
ip t
Cellulose
Page 18 of 18
