Mirjam A. Kabel

Hydrothermally treated xylan rich by-products yield different classes of xylo-oligosaccharides

Four xylan rich by-products, namely wheat bran, brewerys spent grain, corn cobs and Eucalyptus wood, were characterised and subjected to a mild hydrothermal treatment in order to release and degrade the xylan from the starting materials. The chemical characterisation of the feedstock materials, with emphasis on the extracted xylan fractions and using enzymatic degradation of these xylans, resulted in rather detailed pictures of the xylans present. Depending on the feedstock material studied, the xylan present was substituted with arabinose, 4-O-methylglucuronic acid and acetyl groups. During the hydrothermal treatment, arabinose was rather easily removed from the xylan-backbone (wheat bran, brewerys spent grain and corn cobs). The acetyl groups were partly released from the feedstocks, becoming available to catalyse the depolymerisation of the xylan. Also, part of the uronic acids were released, mainly during the treatment of Eucalyptus wood. Due to the partial release of the substituents and cleavage of the xylan by the treatment performed, a wide variety of xylo-oligosaccharides with different structural features corresponding to the xylan-structure of the original feedstock were obtained. Xylo-oligosaccharides branched with arabinose were identified in the hydrolysate from brewerys spent grain, while in the hydrolysate of corn cobs and Eucalyptus wood xylo-oligosaccharides substituted with 4-O-methylglucuronic acid were present as well. Additionally, a series of partially acetylated (acidic) xylo-oligosaccharides was identified in the Eucalyptus wood hydrolysate.

Complex xylo-oligosaccharides identified from hydrothermally treated Eucalyptus wood and brewery's spent grain.

Hydrolysates from two hydrothermally treated xylan-rich agrobased materials, Eucalyptus wood and brewerys spent grain were fractionated by anion-exchange chromatography and size-exclusion chromatography. Hereby, several pools were obtained and they were characterised by their sugar composition. Additionally, the oligosaccharides in the pools described were further identified by high-performance anion-exchange chromatography and mass spectrometry. The hydrothermally treated brewerys spent grain resulted in three pools of which two contained relatively high molecular weight xylan, singly and doubly branched with arabinose [XnAm], separated from a pool of xylo-oligosaccharides less branched with arabinose. The fractionation of the hydrothermally treated Eucalyptus wood resulted in a neutral pool, mainly consisting of a series of (acetylated) xylo-oligosaccharides [XnAcm], and three acidic pools. Two of these acidic pools contained a series of (acetylated) xylo-oligosaccharides including one 4-O-methylglucuronic acid [Xn(GlcAme)1Acm], while the third acidic pool contained (acetylated) xylo-oligosaccharides substituted with two 4-O-methylglucuronic acids [Xn(GlcAme)2Acm]. Additionally, a series of xylo-oligosaccharides containing both 4-O-methylglucuronic acid and a hexose, most likely galactose, was detected in the acidic Eucalyptus pools [Xn(GlcAme)1 or 2AcmH]. Information was obtained about the number of acetyl-groups linked to the (4-O-methylglucurono-) xylo-oligosaccharides. Finally, it is demonstrated with an example that the different substituents to the xylo-oligosaccharides present are of relevance for the fermentability of the xylo-oligosaccharides by human faecal samples.

Characterization of Oligomeric Xylan Structures from Corn Fiber Resistant to Pretreatment and Simultaneous Saccharification and Fermentation

Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans.

A Brief and Informationally Rich Naming System for Oligosaccharide Motifs of Heteroxylans Found in Plant Cell Walls

The one-letter code system proposed here is a simple method to accurately describe structurally diverse oligosaccharides derived from heteroxylans. Substitutions or ‘molecular decoration(s)’ of main-chain d-xylosyl moieties are designated by unique letters. Hence, an oligosaccharide is described by a series of single letters, beginning with the non-reducing d-xylosyl unit. Superscripted numbers are used to indicate the linkage position(s) of main-chain substitution(s) and, where necessary, superscripted lowercase letter(s) indicate the nature of non-glycosidic groups (e.g., methyl, acetyl, or phenolic derivative moieties) that can be present on the substituents. Although relatively simple and practical to use, this abbreviated system lends itself to the naming of a large number of different combinations of structural building blocks and substituents. In its present state, this system is, therefore, adequate to name and differentiate all currently known complex oligosaccharides derived from heteroxylans and is sufficiently flexible to accommodate new structures as they become available.

CORN FIBER, COBS AND STOVER: ENZYME-AIDED SACCHARIFICATION AND CO-FERMENTATION AFTER DILUTE ACID PRETREATMENT

Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached.

Mass determination of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry following HPLC, assisted by on-line desalting and automated sample handling

Abstract The off-line coupling of analytical high-performance anion-exchange chromatography (HPAEC) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. The system was applied to the analysis of neutral and acidic xylo-oligosaccharides. For MALDI-TOF MS, on-line desalting of the HPAEC eluent was performed using an anion self regenerating suppressor (ASRS) in series with a cation self-regenerating suppressor (CSRS). The ASRS permitted the exchange of acetate ions with hydroxide ions while the CSRS permitted the exchange of sodium ions with hydronium ions. The continuous desalting of the eluent was achieved by the electrolysis of pure water in both suppressors. Following automated fractionation after HPAEC separation using a 96-well plate fraction collector and computer-controlled MALDI-TOF MS sample preparation using a robot are applied as well. The complete process from HPAEC separation at analytical scale to MALDI-TOF MS could be performed most conveniently, giving molecular mass information overcoming the rather unpredictable HPAEC elution behavior of (unknown) oligosaccharides.

A generic model for glucose production from various cellulose sources by a commercial cellulase complex

The kinetics of cellulose hydrolysis by commercially available Cellubrix were described mathematically, with Avicel and wheat straw as substrates. It was demonstrated that hydrolysis could be described by three reactions: direct glucose formation and indirect glucose formation via cellobiose. Hydrolysis did not involve any soluble oligomers apart from low amounts of cellobiose. Phenomena included in the mathematical model were substrate limitation, adsorption of enzyme onto substrate, glucose inhibition, temperature dependency of reaction rates, and thermal enzyme inactivation. In addition, substrate heterogeneity was described by a recalcitrance constant. Model parameters refer to both enzyme characteristics and substrate-specific characteristics. Quantitative model development was carried out on the basis of Avicel hydrolysis. In order to describe wheat straw hydrolysis, wheat straw specific parameter values were measured. Updating the pertinent parameters for wheat straw yielded a satisfactory description of wheat straw hydrolysis, thus underlining the generic potential of the model.

Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost.

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained.

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.

Characterisation of cell wall polysaccharides from rapeseed (Brassica napus) meal

To enable structural characteristics of individual cell wall polysaccharides from rapeseed (Brassica napus) meal (RSM) to be studied, polysaccharide fractions were sequentially extracted. Fractions were analysed for their carbohydrate (linkage) composition and polysaccharide structures were also studied by enzymatic fingerprinting. The RSM fractions analysed contained pectic polysaccharides: homogalacturonan in which 60% of the galacturonic acid residues are methyl-esterified, arabinan branched at the O-2 position and arabinogalactan mainly type II. This differs from characteristics previously reported for Brassica campestris meal, another rapeseed cultivar. Also, in the alkali extracts hemicelluloses were analysed as xyloglucan both of the XXGG- and XXXG-type decorated with galactosyl, fucosyl and arabinosyl residues, and as xylan with O-methyl-uronic acid attached. The final residue after extraction still contained xyloglucan and remaining (pectic) polysaccharides next to cellulose, showing that the cell wall matrix of RSM is very strongly interconnected.