Tetramethylammonium hydroxide thermochemolysis for the

85 retention times in gas chromatography of their thermochemolysis derivatives. Despite 86 unavoidable matrix effects, this off-line preparative therm...

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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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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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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

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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

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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

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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

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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

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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

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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

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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

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information about recent organic matter decomposition but also on past depositional

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environments.

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283 5. Conclusion

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When compared with the widely used acid hydrolysis developed for carbohydrates

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analysis, the application of TMAH thermochemolysis to our sediment samples did not

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analyse hemicellulosic carbohydrates and discriminate each individual carbohydrates.

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However, TMAH thermochemolysis allows the analysis of a cellulose pool hidden to acid

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hydrolysis and the specific analysis of free and terminal carbohydrates. As a consequence,

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we caution against making simple direct comparisons of thermochemolysis data with data

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generated from acid hydrolysis because of the different mechanisms involved in each

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process. Nevertheless, and because of their differences, they would be viewed and used as

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pertinent complementary methods for the analysis of carbohydrates in complex

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environmental systems.

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0

of

wood

by

ip t

cr

Characterisation

C Abundance, Solid-

pyrolysis

us

Challinor,

C NMR and IR

derivatisation—gas

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ed

M

an

J.M.

13

13

Page 12 of 18

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cryo-MEB haute resolution, in: F. Elsass F, A.M. Jaunet (Eds.), Structure et ultrastructure des

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407

factors in late stages of forest litter decomposition, Pedobiologia 30 (1987) 101-112.

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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

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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

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cr

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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

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 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

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M

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cr

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Cellulose

Page 18 of 18