The extraction and characterization of soil polysaccharide by whole soil methylation

A soil of the Countesswells series was repeatedly methylated by the Hakomori procedure and a chloroform-soluble product isolated after each methylation. Ninety-two per cent of the material engendered by seven methylations was released during the first four methylations. This had a methoxyl content of about 20% and contained 2% N. Later fractions had lower methoxyl and N contents. Residual carbohydrate in the soil had reducing sugar content on hydrolysis, equivalent to about 3% of the original value. The extracted material had the infrared spectrum of a methylated soil polysaccharide and, on hydrolysis by 2 m trifluoracetic acid, released methylated sugar derivatives of which 68 were characterised by GC-MS. Derivatives corresponding to (1→4) linked sugars predominated for both hexose and pentose sugars but there was also a large proportion of (1→3) linkages, (1→3) linkages predominated for the deoxyhexose sugars. There were more sugars with only one or two methylated hydroxyl groups than could be accounted for as branching points because of the relatively small numbers of end groups. Prior reduction of the soil with sodium borohydride had no measurable effect on the nature or yield of the methylated product. This indicates that amino acid sugar linkages susceptible to β-elimination reactions can have only a very small influence on the reaction. The isolated sugar derivatives accounted for 70% of the total soil sugars. The methylated material before hydrolysis had a low nominal molecular weight on diafiltration, with 68% < 10000. Some of the sugars unaccounted for were probably lost during the dialysis stage necessary to remove dimethyl sulphoxide.     

Structure of “unknown” soil nitrogen investigated by analytical pyrolysis

Curie-point pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and in-source pyrolysis-field ionization mass spectrometry (Py-FIMS) were applied for the first time to the structural characterization of organic nitrogen in hydrolyzates and hydrolysis residues resulting from the classical 6M HCl hydrolysis of mineral soils. Two well-described soils of widely different origin (i.e., a Gleysol Ah and a Podzol Bh) were investigated. Py-GC/MS was performed using a nitrogen-selective detector to detect and identify N-containing pyrolysis in the hydrolyzate (e.g., pyrazole and/or imidazole, N,N-dimethylmethanamine, benzenacetonitrile, propane- and propenenitriles) and the hydrolysis residue (e.g., pyrroles, pyridines, indoles, N-derivatives of benzene, benzothiazol, and long-chain aliphatic nitriles). Moreover, temperature-resolved Py-FIMS allowed us to record the thermal evolution of the N-containing compounds during pyrolysis. These were characterized by a particularly high thermostability compared to their thermal release from whole soils. The combination of pyrolysis with mass spectrometric methods permitted analyses of the identities and thermal stabilities of complex nitrogen compounds in hydrolysis residues of whole soils, which cannot be done by wet-chemical methods. Pyrolysis-methylation GC/MS with tetramethyl-ammonium hydroxide (TMAH) as methylating agent enabled the identification of N,N-dimethylbenzenamine and so confirmed the identification of benzeneamine by Py-GC/MS in nonmethylated hydrolysis residues. The described N-derivatives of benzene and long-chain nitriles are usually not detectable by pyrolysis-mass spectrometry of plants and microorganisms. These compounds are characteristic of soils, terrestrial humic substances and hydrolysis residues and seem to be specific, stable transformation products of soil nitrogen. Received: 15 April 1996     

PHYSICO-CHEMICAL STUDIES ON ENZYME-DEGRADED FULVIC ACID

The enzyme pronase released seven amino acids and also galactose and arabinose from fulvic acid. Hemicellulase released sugars and some amino acids. Removal of the amino acids lowered the molecular weight and gave a lower value for the C. E. C. Differential enzymic removal of carbohydrate and amino acids from the fulvic acid molecule reduced its viscosity. From this evidence it is concluded that the sugar and amino acids are linked to the aromatic core of fulvic acid.     

TITRATIONS OF FULVIC ACID FRACTIONS II: CHEMICAL CHANGES AT HIGH PH

The stability in solution of a polycarboxylic acid fraction and a polysaccharide fraction of fulvic acid was investigated by pH stat titrations. At pH 8.0 alkali-consuming reactions occurred on the polycarboxylic acid fraction. At pH 10.0, these alkali consuming reactions were continuing steadily after 22 days. It is suggested that hydroxylation of bound Al may contribute significantly to the consumption of alkali by the fraction. The data indicated that alkali consuming reactions occurred on the polysaccharide fraction at pH 10.0, but not at pH 9.0 and below. Evidence is discussed which indicates that a ‘peeling process’, by which end reducing sugars are stripped off polysaccharide chains and transformed to saccharinic acids, could account for the alkali consumption on this fraction.     

Effect of periodate treatment of soil on carbohydrate constituents and soil aggregation

Soil from a field under long-term grass was treated with 0.02m sodium periodate for various periods up to 1176 h, followed by 0.1 d sodium tetraborate for 6 h. This destroyed an increasing proportion of microaggregates >45 μm and carbohydrate. After periodate treatment for 6 h about 70% of the soil sugars remained in the residue as measured by reducing sugar content and about 67% as individual sugars measured by gas-liquid chromatography. After 48 h the reducing sugar content was about 45%. An inverse linear relationship was established between the proportion by weight of microaggregates >45 μm and residual carbohydrate. The residual carbohydrate showed an enrichment in sugars commonly found in plant materials; glucose, arabinose and xylose, suggesting that the microbial carbohydrate had been preferentially destroyed. When the concentration of the periodate was increased to 0.05 m the residue contained about 50% of the original carbohydrate after 6 h treatment, and 25% after 48 h and an additional increase of about 10% in the proportion by weight of particles in the <45 μm range. These results throw doubt on the validity of assumptions made in a number of studies about the limited extent to which soil polysaccharide is involved in aggregation.     

On the components of soil humic acids (part 9)

The carbohydrates in soil organic matter seem to be derived from undecomposed or partially decomposed plant and microbial residues, In soil, these carbohydrates exist chiefly in such from as polysaccharide hemicellulose, and their polyuronide has been the chief object of investigation^1)-3). In various soil, the polyuronide is found in a large quantity in fulvic fraction of soil organic matter and has been considered as important in connection with the physical structure of soil^4),5). According to Lynch ⁵⁾, the carbohydrate content of humic acid is markedly smaller than that of fulvic acid. His work also indicates that a' considerable change is noted in the content and composition of the carbohydrate in humic acid because of the addition of some organic substances to the soils, or of the cultivation of virgin soils. Further, some investigators⁷⁾ believe that uronic acid is introduced into the aromatic structure of the humic acid by changing into pentose and furan. Accordingly, it seems that the role of carbohydrate in the formation of soil humic acid should not be overlooked.     

Chemical composition of the organic matter in forest soils: The humus layer

Decomposition and humification were studied within three types of forest humus (mull, moder, and mor) by means of CPMAS ¹³C NMR spectroscopy combined with degradative methods. The NMR data show that O-alkyl carbon decreases in all soils, and alkyl as well as carboxyl carbon increase as depth and decomposition increase; the percentage of aromatic carbon remains constant at about 25%. With increasing depth the amount of carbon that can be identified as belonging to specific compound classes by wet chemical methods decreases from 60% to 40%. Microbial polysaccharides and the proportion of non polysaccharide O-alkyl carbon increase with depth. A selective preservation of recalcitrant, condensed lignin structural units is also observed. In order to relate the spectroscopic and chemical data from investigations of whole soils with studies of humification, samples were fractionated into fulvic acid, humic acid, and humin fractions. The fulvic acid fraction contains large concentrations of carbohydrates irrespective of the soil horizon. The humic acid fraction contains less polysaccharides, but high amounts of alkyl carbon and aromatic structures. The percentage of aromatic carbon existing in the humic acid fraction increases with depth, probably reflecting the amount and degree of oxidative decomposition of lignin. A loss of methoxyl and phenolic groups is evident in the ¹³C NMR spectra of the humic acid fraction. The humin fraction resembles relatively unchanged plant-derived materials as evident from the lignin parameters and carbohydrate contents. All the observed data seem to indicate that humic acids originate form oxidative degradation of humin or plant litter.     

Sequential exhaustive extraction of a Mollisol soil, and characterizations of humic components, including humin, by solid and solution state NMR

A comprehensive sequential extraction procedure was applied to isolate soil organic components using aqueous solvents at different pH values, base plus urea (base‐urea), and finally dimethylsulfoxide (DMSO) plus concentrated H₂SO₄ (DMSO‐acid) for the humin‐enriched clay separates. The extracts from base‐urea and DMSO‐acid would be regarded as ‘humin’ in the classical definitions. The fractions isolated from aqueous base, base‐urea and DMSO‐acid were characterized by solid and solution state NMR spectroscopy. The base‐urea solvent system isolated ca. 10% (by mass) additional humic substances. The combined base‐urea and DMSO‐acid solvents isolated ca. 93% of total organic carbon from the humin‐enriched fine clay fraction (<2 μm). Characterization of the humic fractions by solid‐state NMR spectroscopy showed that oxidized char materials were concentrated in humic acids isolated at pH 7, and in the base‐urea extract. Lignin‐derived materials were in considerable abundance in the humic acids isolated at pH 12.6. Only very small amounts of char‐derived structures were contained in the fulvic acids and fulvic acids‐like material isolated from the base‐urea solvent. After extraction with base‐urea, the 0.5 m NaOH extract from the humin‐enriched clay was predominantly composed of aliphatic hydrocarbon groups, and with lesser amounts of aromatic carbon (probably including some char material), and carbohydrates and peptides. From the combination of solid and solution‐state NMR spectroscopy, it is clear that the major components of humin materials, from the DMSO‐acid solvent, after the exhaustive extraction sequence, were composed of microbial and plant derived components, mainly long‐chain aliphatic species (including fatty acids/ester, waxes, lipids and cuticular material), carbohydrate, peptides/proteins, lignin derivatives, lipoprotein and peptidoglycan (major structural components in bacteria cell walls). Black carbon or char materials were enriched in humic acids isolated at pH 7 and humic acids‐like material isolated in the base‐urea medium, indicating that urea can liberate char‐derived material hydrogen bonded or trapped within the humin matrix.     

New method for a two-step hydrolysis and chromatographic analysis of pectin neutral sugar chains

A new method for the determination of the main neutral sugars in pectin has been developed. The sample preparation involves a mild chemical attack followed by an enzymatic hydrolysis. The completeness and nondestructive character of the method are demonstrated by comparison of the results obtained with different acids such as H2SO4, HCl, and trifluoroacetic acid (TFA) at different concentrations (2, 1, or 0.2 M) at two temperatures (80 or 100 degrees C). The chemical hydrolysis of pectin neutral sugar chains with strong acid (1 or 2 M) and high temperature (100 degrees C) shows that the liberation of the pectin sugars is not realized at the same rate for each sugar. Different optimum conditions are thus obtained. However, the chemical pectin hydrolysis with 0.2 M TFA at 80 degrees C is characterized by the liberation of pectin neutral sugar side chains without any degradation within 72 h of hydrolysis. Under these conditions, the liberation of some pectin sugars, essentially galactose, glucose, and rhamnose, was not complete. An enzymatic hydrolysis is necessary to obtain a complete release of all the sugars. The combination of the two treatments, a chemical hydrolysis realized with diluted acid (0.2 M) for 72 h at low temperature (80 degrees C) on one hand and an enzymatic hydrolysis on the other hand, allow a total liberation of pectin sugars. The quantitative analysis of the carbohydrates is realized with accuracy, high selectivity, and sensitivity with high-performance anion-exchange chromatography with pulsed-amperometric detection. The sugars can be analyzed without any derivatization with a limit of quantification of 0.1 mM.     

ORIGINS AND STABILITY OF SOIL POLYSACCHARIDE

Recent work on the origin of soil polysaccharide and its biological stability in soil is reviewed. It is concluded that much of the constituent hexose and deoxyhexose sugars are of microbial origin, whereas the pentose sugars are derived from plant residues. The stability of soil polysaccharide in its native state is not related to its chemical composition but to its unavailability. This is caused by inaccessibility within undecomposed biological residues and to insolubility resulting from adsorption on clay, the formation of metal complexes, or tanning by soil humic substances. Complexing by metals and tanning may also inhibit enzymic hydrolysis.     

Compound‐specific stable‐isotope (δ13C) analysis in soil science

This review provides current state of the art of compound‐specific stable‐isotope‐ratio mass spectrometry (δ¹³C) and gives an overview on innovative applications in soil science. After a short introduction on the background of stable C isotopes and their ecological significance, different techniques for compound‐specific stable‐isotope analysis are compared. Analogous to the δ¹³C analysis in bulk samples, by means of elemental analyzer–isotope‐ratio mass spectrometry, physical fractions such as particle‐size fractions, soil microbial biomass, and water‐soluble organic C can be analyzed. The main focus of this review is, however, to discuss the isotope composition of chemical fractions (so‐called molecular markers) indicating plant‐ (pentoses, long‐chain n‐alkanes, lignin phenols) and microbial‐derived residues (phospholipid fatty acids, hexoses, amino sugars, and short‐chain n‐alkanes) as well as other interesting soil constituents such as “black carbon” and polycyclic aromatic hydrocarbons. For this purpose, innovative techniques such as pyrolysis–gas chromatography–combustion–isotope‐ratio mass spectrometry, gas chromatography–combustion–isotope‐ratio mass spectrometry, or liquid chromatography–combustion–isotope‐ratio mass spectrometry were compared. These techniques can be used in general for two purposes, (1) to quantify sequestration and turnover of specific organic compounds in the environment and (2) to trace the origin of organic substances. Turnover times of physical (sand < silt < clay) and chemical fractions (lignin < phospholipid fatty acids < amino sugars ≈ sugars) are generally shorter compared to bulk soil and increase in the order given in brackets. Tracing the origin of organic compounds such as polycyclic aromatic hydrocarbons is difficult when more than two sources are involved and isotope difference of different sources is small. Therefore, this application is preferentially used when natural (e.g., C3‐to‐C4 plant conversion) or artificial (positive or negative) ¹³C labeling is used.     

Interpretation of heteronuclear and multidimensional NMR spectroscopy of humic substances

Mapping the chemical structures and organization of humic substances is vital for a fundamental understanding of their roles and interactions in the soil. One‐dimensional nuclear magnetic resonance (NMR) techniques have advanced our awareness of the composition of humic materials, but modern developments in two‐dimensional NMR are soon likely to make obsolete reliance on one‐dimensional spectra alone. The advantages of using heteronuclear two‐dimensional NMR spectroscopy are illustrated in this paper in studies of the structural units in a fulvic acid fraction isolated from the Bh horizon of a Podzol. The structures identified from the NMR (at 500 MHz) experiments can be summarized as: mono‐ and dicarboxylic acids (in about equal amounts), with an average chain length of about 10 carbon atoms (these are easily the major components); smaller amounts (about 10–20% of the acids) of esters and alcohols or ethers; some carbohydrate and amino acid residues (evidence from chemical shift data would suggest that these were likely to be in the form of chains); and very small amounts of 1,2‐, 1,4‐, and 1,3,4‐substituted benzenes and of cinnamic acids. The results suggest that applications of heteronuclear and multidimensional NMR spectroscopy will allow considerable progress to be made in understanding the nature of the structural units and their connectivities in humic molecules provided that the heterogeneity of the humic mixtures can first be decreased significantly.     

日本常规耕作和有机耕作(翻耕与免耕)农田的动物区系(螨和线蚓)比较

Nitrogen forms of humic substances from a subalpine meadow soil,a lateritic red soil and a weathered cola and the effect of acid hydrolysis on N structures of soil humic substances were studied by using ^15N cross-polarization magic angle spinning nuclear magnetic resonance(CPMAS NMR) spectroscopy,Of the detectable ^15N-signal intensity in the spectra of soil humic substances 71%-79% may be attributed to amide groups ,10%-18% to aromatic/aliphatic amines and 6%-11% to indole-and pyrrole-like N.Whereas in the spectrum of the fulvic acid from weathered coal 46%,at least,of the total ^15N-signal intensity might be assigned to pyrrole-like N,14% to aromatic/aliphatic amines,and the reamining intensities could not be assigned with certainty,Data on nonhydrolyzable reside of protein-sugar mixture and a ^15N-labelled soil fulvic acid confirm the formation of nonhydrolyzable heterocyclic N during acid hydrolysis.     

Characterization of sinapyl derivatives in pineapple (Ananas comosus [L.] Merill) juice.

Three previously unidentified phenolic compounds were found in pineapple (Ananas comosus [L.] Merill) juice in substantial concentrations and were isolated by semipreparative reverse phase HPLC. The structures were elucidated from UV spectra, acid hydrolysis, and subsequent amino acid analysis, mass spectrometry, and two-dimensional NMR spectroscopy. The compounds are identified as S-sinapyl-L-cysteine, N-L-gamma-glutamyl-S-sinapyl-L-cysteine, and S-sinapylglutathione.     

Fulvic acid studies: evidence for a polycarboxylate co-ordination mode at soil pH

The reactions of two fulvic acid samples with Cu²⁺ and Cd²⁺ have been studied by potentiometric titration (pH and ion selective electrode). The metal-uptake curves for samples initially rendered ‘metal-free’ by a precipitation-solvent extraction technique resemble those for citric and malic acids at soil pH (4–6.5). The presence of aliphatic and aromatic components was established by ¹³C nmr spectroscopy; the presence of phenolic components was inferred from pH titrations. It is shown that a polycarboxylate component (simulated by citrate) would mask reactions of phenolic components with metals in weakly acidic and neutral media. The mode of coordination between fulvic acid and metals will vary with pH and with the metal: fulvic acid ratio.     

氮肥和羊粪对内蒙古典型草原土壤多糖含量及组成的影响

采用三氟乙酸(TFA)水解、糖醇乙酸酯衍生、气相色谱法测定土壤多糖含量,研究了内蒙古羊草草原围栏封育并连续5年施用氮肥和羊粪的表层土壤的多糖含量和组成特征。结果表明:长期施用氮肥显著降低土壤多糖含量6%～19%;施用羊粪显著提高土壤多糖含量20%。施氮肥或羊粪都降低了(半乳糖+甘露糖)(/阿拉伯糖+木糖)(GM/AX)和甘露糖/木糖(M/X)的比例,表明施肥降低了微生物多糖对土壤多糖的贡献,但是施氮肥的土壤降低幅度大于施羊粪的土壤。这表明,长期施氮肥和羊粪都将改变土壤多糖含量和组成。     

Galactoglucomannan Oligosaccharides (GGMO) from a molasses byproduct of pine ( Pinus taeda ) fiberboard production

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a β-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.     

A simple wet chemical extraction procedure to characterize soil organic matter (Som): 3. Results of vegetation,crop litter,and forest litter in comparison to data as revealed with CPMAS 13NMR spectroscopy and pyrolysis‐field ionization mass spectroscopy

Abstract

Data on the organic matter composition of vegetation, crop litter, and forest litter of Oi (=L) horizons from several European locations available with the litter compound analysis (LCA) discussed in previous communications are presented. The wet chemical results are discussed in comparison to data as revealed by cross polarization magic angle spinning carbon‐13 nuclear magnetic resonance spectroscopy (CPMAS ¹³C‐NMR) and pyrolysis‐field ionization mass spectrometry (Py‐FIMS). The LCA of vegetation, crop litter, and forest litter provides much detailed information about the chemical composition of extractable organic matter. Normally, both CPMAS ¹³C‐NMR and Py‐FIMS confirm the wet chemical results. Our data suggest a moderate chemical variation between the fresh organic residue and the litter. NMR revealed structural information about the non‐extractable organic matter using a combination of wet chemical extraction and CPMAS ¹³C‐NMR of whole soil. In addition, Py‐FIMS identified specific SOM segments at a qualitative level.     

STRUCTURAL STUDIES ON SOIL POLYSACCHARIDE

The polysaccharide extracted by alkali from a Countesswells series soil has been fully methylated and the hydrolysis products identified by GC-MS. The parent neutral sugars are galactose, glucose, mannose, arabinose, xylose, fucose and rhamnose and these constitute about 40 per cent of the polysaccharide. The analysis shows that hexose components are predominantly present in 1 → 3 and 1 → 4 linkages and pentose sugar in 1 → 4 linkages. About 20 per cent of the residues were in branching positions. From the number of non-reducing terminal groups present the average molecular weight of the methylated material has been calculated to be about 1460 compared with a value of 2700 obtained by vapour pressure osmometry. This contrasts with much higher values reported for unmethylated soil polysaccharides. The mixture of derivatives obtained supports the concept that soil polysaccharide originates in both plants and microorganisms.     

Microbial residues as indicators of soil restoration in South African secondary pastures

Prolonged intensive arable cropping of semiarid grassland soils in the South African Highveld resulted in a significant loss of C, N and associated living and dead microbial biomass. To regenerate their soils, farmers converted degraded arable sites back into secondary pastures. The objective of this study was to clarify the contribution of microorganisms to the sequestration of C and N in soil during this regeneration phase. Composite samples were taken from the topsoils of former arable land, namely Plinthustalfs, which had been converted to pastures 1-31 years ago. Amino sugars were determined as markers for microbial residues in the bulk soil and in selected particle-size fractions. The results showed that when C and N contents increased during the secondary pasture usage, the amino sugar concentration in the bulk soil (0-5 cm) recovered at similar magnitude and reached a new steady-state level after approximately 90 years, which corresponded only to 90% of the amino sugar level in the primary grassland. The amino sugar concentration in the clay-sized fraction recovered to a higher end level than in the bulk soil, and also at a faster annual rate. This confirms that especially the finer particles contained a high amount of amino sugars and were responsible, thus, for the restoration of microbially derived C and N. The incomplete recovery of amino sugars in bulk soil can only in parts be attributed to a slightly coarser texture of secondary grassland that had lost silt through wind erosion. The soils particularly had also lost the ability to restore microbial residues below 5 cm soil depth. Overall, the ratios of glucosamine to muramic acid also increased with increasing duration of pasture usage, suggesting that fungi dominated the microbial sequestration of C and N whereas the re-accumulation of bacterial cell wall residues was less pronounced. However, the glucosamine-to-muramic acid ratios finally even exceeded those of the primary grassland, indicating that there remained some irreversible changes of the soil microbial community by former intensive crop management.