Elsevier

Organic Geochemistry

Volume 41, Issue 6, June 2010, Pages 580-585
Organic Geochemistry

Analysis of hydrolysable neutral sugars in mineral soils: Improvement of alditol acetylation for gas chromatographic separation and measurement

https://doi.org/10.1016/j.orggeochem.2010.02.009Get rights and content

Abstract

To understand plant–microbe relationships, a simple method is required for identification of the nature of soil polysaccharides. Acid hydrolysis, reduction of sugar monomers to the corresponding alcohols and subsequent derivatisation with acetic anhydride prior to gas chromatography has often been used for identification and quantification of hydrolysable sugars in plant and soil samples. In mineral soil samples, precipitation of iron hydroxides and dissolved organic substances after addition of ammonia may lead to co-precipitation of the analytes, leading to an underestimation of the neutral sugar content. The aim of this study was to adapt the derivatisation procedure for soil samples with large iron (hydr)oxide contents. This was done by omission of ammonia and addition of ethylenediaminetetraacetic acid (EDTA) to keep iron in solution and to avoid co-precipitation. Standard addition approaches show that the recovery of all sugars is enhanced with the modified method. Application of the EDTA method improves the recovery of added internal standard, increases yields of sugars in mineral soils and reduces the observed standard error compared to the ammonia method. This was shown for a set of various soil samples with different iron (hydr)oxide contents. The EDTA method is also applicable for mineral free samples and therefore suitable for routine use.

Introduction

Sugars account for 50% of plant litter entering the soil system (Kögel-Knabner, 2002). Carbohydrates represent about one quarter of soil organic matter, much of it being derived from plant polysaccharides in roots and plant debris (Oades, 1984). Soil polysaccharides derive not only from plants, but likewise from animal and microbial products and are resynthesised by soil organisms (Cheshire et al., 1973). Microaggregates (<250 μm) are mainly stabilised by the binding action of polysaccharides produced by bacteria, but also by plant roots and fungal hyphae (Oades, 1984). Water stable aggregates are enriched in polysaccharides of microbial origin compared to unstable ones. This can be explained by the fact that aggregate stability is mediated partly by extracellular polysaccharides of microorganisms developing on plant debris occluded within the aggregates (Puget et al., 1999). Therefore, there is not only interest in the polysaccharide content, but also in the origin of the sugars. Amino sugars are generally not found in plants and can be used as biomarker for microbial soil communities (Bodé et al., 2009). Neutral sugars are used to distinguish between plant derived and microbial derived polysaccharides, which can be estimated by the ratio of (galactose + mannose)/(arabinose + xylose) [GM/AX] (Oades, 1984). Plant materials contain substantial quantities of pentose sugars, mainly arabinose and xylose, resulting in small GM/AX ratios (<0.5) for plant polysaccharides. Soil microorganisms synthesise dominantly galactose and mannose (hexose sugars), resulting in large GM/AX ratios (>2.0) for microbial polysaccharides (Oades, 1984).

A gas chromatographic (GC) analysis method for hydrolysable polysaccharides in forest floor layers and mineral soil horizons as alditol acetate derivatives of their monosaccharides was first described by Spiteller (1980). However, this procedure required more than two days of sample preparation prior to GC analysis. In the following two decades, modifications concentrated on complex organic matrices (Kögel and Bochter, 1985, Black and Fox, 1996, Fox, 1999), including substantially improved chemical clean up steps, both before and after derivatisation. Each stage of the procedure (hydrolysis, pre-derivatisation clean up, reduction, acetylation and post-derivatisation clean up) has been optimised to decrease background noise and allow analysis of large batches of samples.

First improvements and simplifications for the analysis of sugar monomers released from mineral soil samples using reduction acetylation were described by Rumpel and Dignac (2006). Their method basically consists of the following steps: trifluoroacetic acid (TFA) hydrolysis according to Amelung et al. (1996), reduction of the monomers to the corresponding alcohols with NaBH4, derivatisation with acetic anhydride, gas chromatographic separation and detection with flame ionisation detector (FID). When applied to mineral soil samples, dissolved iron (hydr)oxides are precipitated from the hydrolysate by ammonia addition (Rumpel and Dignac, 2006). However, these precipitates may cause co-precipitation of dissolved organic compounds including neutral sugar monomers (Kalbitz and Kaiser, 2008), especially when applied to subsoil horizons as observed for B horizons of a Cambisol (Rumpel and Dignac, 2006).

The aim of this study was to modify the method of Rumpel and Dignac (2006) to make it applicable to mineral soil samples with large iron (hydr)oxide contents by avoiding precipitation of iron hydroxides. Thus, we tested an alternative approach, using ethylenediaminetetraacetic acid (EDTA) which keeps iron ions in stable, soluble complexes.

Section snippets

Soils

A range of soil samples differing in land use, regional provenance and iron (hydr)oxide content were used. Basic properties of all soil samples are given in Table 1. Soils were classified according to IUSS Working Group Reference Base, 2006. All samples were air dried, sieved to <2 mm and ground before sugar analysis.

Samples with lowest contents in iron (hydr)oxides are the Ha horizon and the He horizon derived from Hemic Histosols (Dystric) sampled at the Fichtelgebirge, Germany (Prietzel et

Observations during pre-treatment and derivatisation

The addition of ammonia after hydrolysis of mineral soil samples leads to the formation of green precipitates. Precipitation occurred only with mineral soil samples rich in iron (hydr)oxides (e.g. Bw and Bs horizons), not with organic samples (e.g. Ha and He horizons). These precipitates are assumed to be green rust that forms in consequence of an increased pH, identified by their change in colour after contact with oxygen into a deep ochre to rusty red brown (Cornell and Schwertmann, 2003). If

Summary and conclusions

Our study introduced the replacement of ammonia by EDTA for the preparation of neutral sugar monomers before alditol acetate derivatisation. This resulted in gas chromatograms without confounding peaks which reduce the quality of the separation. Yields of neutral sugars and recoveries as well as the precision of determination are increased. Omission of ammonia and addition of EDTA avoids the formation of iron hydroxide precipitates and subsequent neutral sugar losses caused by co-precipitation,

Acknowledgements

We gratefully acknowledge Samuel Bodé and an anonymous reviewer who provided helpful comments on the manuscript.

References (21)

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