Manuel Becker

Severe drought stress is affecting selected primary metabolites, polyphenols, and volatile metabolites in grapevine leaves (Vitis vinifera cv. Pinot noir)

Extreme weather conditions with prolonged dry periods and high temperatures as well as heavy rain events can severely influence grapevine physiology and grape quality. The present study evaluates the effects of severe drought stress on selected primary metabolites, polyphenols and volatile metabolites in grapevine leaves. Among the 11 primary metabolites, 13 polyphenols and 95 volatiles which were analyzed, a significant discrimination between control and stressed plants of 7 primary metabolites, 11 polyphenols and 46 volatile metabolites was observed. As single parameters are usually not specific enough for the discrimination of control and stressed plants, an unsupervised (PCA) and a supervised (PLS-DA) multivariate approach were applied to combine results from different metabolic groups. In a first step a selection of five metabolites, namely citric acid, glyceric acid, ribose, phenylacetaldehyde and 2-methylbutanal were used to establish a calibration model using PLS regression to predict the leaf water potential. The model was strong enough to assign a high number of plants correctly with a correlation of 0.83. The PLS-DA provides an interesting approach to combine data sets and to provide tools for the specific evaluation of physiological plant stresses.

Ethoximation-silylation approach for mono- and disaccharide analysis and characterization of their identification parameters by GC/MS.

The qualitative and quantitative analysis of complex carbohydrate mixtures is a challenging problem. When tackled by GC/MS, close retention times and largely similar mass spectra with no specific features complicate unambiguous identification, especially of monosaccharides. An optimized pre-capillary ethoximation-silylation GC/MS method for determination of monosaccharides and disaccharides was applied to a wide range of analytes (46 compounds). The two-step derivatization resulted in a pair of syn and anti peaks with specific retention and intensity ratio. The resulting dataset of mass spectra was subjected to a PCA-based pattern recognition. An oxime peak identifier (OPI) of the carbohydrate analytes, based on the combination of an internal standard and the corresponding syn/anti peak ratios, increased the reliability of the identification of reducing carbohydrates. Finally, the introduced EtOx-TMS derivatization method was applied to four different carbohydrate matrices (agave sirup, maple sirup, palm sugar, and honey).

Determination of carbohydrate- and lignin-derived components in complex effluents from cellulose processing by capillary electrophoresis with electrospray ionization-mass spectrometric detection

Degradation products from lignocellulosic materials receive increasing attention due to the continuously growing interest in their utilization. The inherent structural variance of lignocellulosics combined with the intricacy of lignocellulosic processing (e.g. pulping of wood and bleaching of cellulosic pulps) and the complexity of degradation processes occurring therein result in rather complex mixtures in the process streams and effluents that contain a large quantity of structurally different degradation products. This is true for most processing steps, but also for degradation reactions occurring during aging of lignocellulosic materials, such as paper, cellulosic tissue or textiles. In order to render such mixtures better analytically accessible than hitherto possible a CE-ESI-MS method was established for the simultaneous determination of aliphatic carboxylic acids from the degradation of (hemi)celluloses and aromatic compounds from lignin degradation. CE and ESI-MS parameters have been optimized towards sensitivity and good reproducibility. The method was tested in two real-world scenarios: the determination of major components in effluents from bleaching stages in the pulp and paper industry, and the analysis of degradation products in extracts of naturally aged papers. The advantages and drawbacks of this approach are critically discussed.

A novel method to analyze the degree of acetylation in biopolymers.

A novel approach to measure the degree of acetylation in biopolymers applying a combination of Zemplén-deacetylation by sodium methanolate and GC-MS methodology is introduced. The development focuses on very low limits of detection to cover also samples with extremely low degrees of acetylation which hitherto eluded accurate determination. Free acetic acid or inorganic acetates, often present in biopolymer samples, do not disturb the quantification. Two techniques to measure the Zemplén-released methyl acetate were comparatively assessed, direct injection of the liquid phase and a SPME-based approach, the former being more straightforward, but being inferior to the latter in sensitivity. By applying isotopically labeled methyl acetate released from 4-O-(13C2-acetyl)-vanillin as the internal standard, influences, such as varying moisture contents, are corrected, improving the overall method reliability to a large extent. The combination of Zemplén-release of acetyl groups in biopolymers as methyl acetate, in connection with its accurate quantification by SPME-GC-MS, was found to be the method of choice for routine, yet very accurate analysis of a wide range of acetylation degrees of biopolymers, showing satisfying analytical parameters along with easy handling and widest applicability. Limit of detection for acetylated cellulose samples is 0.09nmol/mg, for hemicellulose samples 0.48nmol/mg.

Degradation products of lignocellulosics in pulp mill effluents - comparison and evaluation of different gas chromatographic techniques for a comprehensive analysis.

Abstract Pulp mill effluents contain potentially valuable compounds and will gain even greater importance than today in future biorefinery scenarios as possible resources. Analysis of effluents of process streams is not straightforward because of the vast variety of substances embedded in the complex inorganic matrix of high concentration. In the present comparative investigation, different combinations of gas chromatography (GC) and derivatisation techniques were tested and critically evaluated aiming at a comprehensive description of all major compound classes (low-molecular weight and long-chain carboxylic acids, lignin fragments, carbohydrates and aldehydes). As derivatisation techniques, trimethylsilylation, (trimethylsilyl)methylation and methylation were applied. In addition, the effluents were analysed by pyrolysis-GC/mass spectrometry (MS), either directly or with simultaneous methylation. The study provides comprehensive information on the composition of effluents, the suitability of the various combinations of derivatisation and GC/MS techniques. The discrimination effects of the different approaches are compared. The comprehensive analytical approach led to a coherent balance in terms of the contribution of the major portion of organic compounds to the total organic carbon (TOC) content in pulp mill effluents.

Side reactions of 4-acetamido-TEMPO as the catalyst in cellulose oxidation systems

Abstract TEMPO/hypochlorite oxidation of cellulosics are frequently used to obtain water-soluble polyglucuronates or nano-disperse materials. The reaction under neutral conditions offers a big advantage over hitherto applied alkaline systems as base-induce β-elimination reactions causing severe cellulose degradation are suppressed. Although 4-acetamido-TEMPO seems to offer high initial activities in cellulose oxidation, the TEMPO derivative is irreversibly converted into species with much lower reactivity. The degradation pathways and the chemical structure of the 4-acetamido-TEMPO-derived (by)products have been clarified. Degradation is strongly dependent on the pH value of the reaction mixture. In acidic conditions (pH<5), 4-oxo-TEMPO is formed by an oxidative deamination process, the 4-oxo-derivative still being capable of performing cellulose oxidation. In alkaline media, a similar process proceeds, but is immediately followed by a Favorskij rearrangement to a pyrrolidine-3-carboxylic acid (PROXYL) derivative. In both cases, the released acetamido moiety is slowly converted into methylamine by a decarboxylative rearrangement (Hofmann degradation). The results suggest that the instability and the chemical fate of 4-acetamido-TEMPO needs to be considered when cellulose oxidations and their kinetics are addressed: the recyclability of the 4-acetamido-TEMPO catalyst is rather limited.