Bodo Saake

Molar mass determination of lignins by size-exclusion chromatography: towards standardisation of the method

Abstract The reactivity and physicochemical properties of lignins are partly governed by their molar mass distribution. The development of reliable standard methods for determination of the molar mass distribution is not only relevant for designing technical lignins for specific applications, but also for monitoring and elucidating delignification and pulping processes. Size-exclusion chromatography (SEC) offers many advantages, such as wide availability, short analysis time, low sample demand, and determination of molar mass distribution over a wide range. A collaborative study has been undertaken within the “Eurolignin” European thematic network to standardise SEC analysis of technical lignins. The high-molar-mass fraction of polydisperse lignins was shown to be the main source of intra- and interlaboratory variations, depending on the gel type, elution solvent, detection mode, and calculation strategy. The reliability of two widespread systems have been tested: one based on alkali and a hydrophilic gel (e.g., TSK Toyopearl gel) and the other based on THF as solvent and polystyrene-based gels (e.g., Styragel). A set of practical recommendations has been deduced.

Enzyme-aided characterisation of carboxymethylcellulose

Two conventionally prepared carboxymethylcelluloses (CMCs) with differing degrees of substitution (DS: 0.6 and 1.2) were fragmented by endoglucanase treatment until no further degradation was possible. This treatment brought about a decisive improvement in the water solubility of the polymers. The degraded samples were separated into 18 fractions by size exclusion chromatography (SEC). The quality of the preparative SEC was monitored by analytical SEC equipped with a multiple detector system. After acidic hydrolysis of each individual fraction, the carboxymethyl derivatives of the anhydroglucose units were determined by anion exchange chromatography and pulsed amperometric detection. The accessibility of endoglucanase action was clearly a function of the degree of substitution (DS). As the DS increased the efficiency of the enzyme became more limited. It could also be demonstrated that the polysaccharide chain of both CMC samples included some regions that were highly substituted and some other regions of low substitution.

A comparison of lignin polymer models (DHPs) and lignins by 31P NMR spectroscopy

Abstract Fractionated guaiacyl (G) and guaiacyl/syringyl (GS) DHPs prepared by continuous (Zutropf, ZT) and discontinuous (Zulauf, ZL) dehydrogenation schemes were subjected to quantitative 31P NMR spectroscopy and their various hydroxyl groups determined. Two milled wood lignin samples from softwood and hardwood species were also examined. It was found that GS-DHPs resemble more GS milled wood lignins than G-DHPs resemble G milled wood lignins. The total phenolic-OH contents of ZT-DHPs are always lower than those of ZL-DHPs, in good agreement with the theory which predicts more β-O-4 linkages in the former. Furthermore, in agreement with the dehydrogenation theory, the bulk character of G-DHPs results from the formation of condensed units. In GS-DHPs, the S units are etherified early in the polymerization process, while the bulk character of such samples is mainly due to G-units. Regarding the secondary hydroxyl groups the results obtained for the G-DHPs are extremely low. This underlines again the principal differences of G-DHPs compared with GS-DHPs and MWLs. The erythro to threo ratio of the G-type samples was found to vary between 1 and 1.5, indicating only minor dependence on molar mass or mode of preparation. The erythro to threo ratio in GS-type samples was found to vary from 1.6 to 4.3, showing the highest value for low molar mass ZL-DHPs and lowest values for the cherry tree MWL.

Methodology for quantitative determination of the carbohydrate composition of brown seaweeds (Laminariaceae)

The monosaccharide composition of four different samples of brown seaweeds Laminaria digitata and Saccharina latissima were compared by different high performance anion exchange chromatography (HPAEC) methods after different acid hydrolysis treatments or a cellulase treatment. A two-step treatment of 72% (w/w) H2SO4 + 4% (w/w) H2SO4 performed best, but cellulase treatment released more glucose than acid treatments. HPAEC with pulsed amperometric detection (PAD) allowed quantification of all present neutral sugars and the sugar alcohol mannitol. Furthermore, the use of guluronic, glucuronic, and galacturonic acid as standards enabled quantification of the uronic acids. A complete map of amino acids, fatty compounds, minerals, and ash was also achieved. L. digitata and S. latissima harvested in Denmark April (Baltic Sea, 2012) were dominated by alginic acid and ash (each ∼30% by weight (w/w) of the dry matter) and 10% (w/w) protein. In contrast, the dominant compound of L. digitata harvested in August (North Sea, 2012) was glucose constituting 51% w/w of the dry matter, and with 16% w/w alginic acid. Washing prior to analysis mainly removed salts.

Studies of xylan interactions and cross-linking to synthetic lignins formed by bulk and end-wise polymerization: a model study of lignin carbohydrate complex formation

The mechanism of lignin carbohydrate complex formation by addition of polysaccharides on quinone methide (QM) generated during lignin polymerisation was investigated using a model approach. Dehydrogenation polymers (DHPs, lignin model compounds) were synthesized from coniferyl alcohol in the presence of a glucuronoarabinoxylan (GAX) extracted from oat spelts, by Zutropfverfahren (ZT) and Zulaufverfahren (ZL) methods. The methods ZT and ZL differed in their distribution of QM over the reaction period but generated roughly the same QM amount. Steric exclusion chromatography of the ZT and ZL reaction products showed that only the ZT reaction produced high molar mass compounds. Covalent linkages in the ZT reaction involving ether bonds between GAX moiety and α carbon of the lignin monomer were confirmed by 13C NMR and xylanase-based fractionation. The underlying phenomena were further investigated by examining the interactions between GAX and DHP in sorption experiments. GAX and DHPs were shown to interact to form hydrophobic aggregates. In the ZT process, slow addition permitted polymer reorganisation which led to dehydration around the lignin-like growing chains thereby limiting the addition of water on the quinone methide formed during polymerisation and thus favoured lignin–carbohydrate complex (LCC) formation.

Optimization of steam pretreatment conditions for enzymatic hydrolysis of poplar wood

Abstract Steam refining was investigated as a pretreatment for enzymatic hydrolysis of poplar wood from a short rotation plantation. The experiments were carried out without debarking to use an economically realistic raw material. Steam refining conditions were varied in the range of 3–30 min and 170–220°C, according to a factorial design created with the software JMP from SAS Institute Inc., Cary, NC, USA. Predicted steaming conditions for highest glucose and xylose yields after enzymatic hydrolysis were at 210°C and 15 min. Control tests under the optimized conditions verified the predicted results. Further pretreatments without bark showed that the enzymes were not significantly inhibited by the bark. The yield of glucose and xylose was 61.9% of theoretical for the experiments with the whole raw material, whereas the yield for the experiments without bark was 63.6%. Alkaline extraction of lignin from the fibers before enzymatic hydrolysis resulted in an increase of glucose yields from mild pretreated fibers and a decrease for severe pretreated fibers. The extracted lignin had a high content of xylose of up to 14% after very mild pretreatments. On the other hand, molecular weights of the extracted lignin increased substantially after pretreatments with a severity factor above 4. Hence, alkaline extraction of the lignin seems only attractive in a narrow range of steaming conditions.

Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families

Cellulose acetate (CA) was found to be a substrate of several acetyl xylan esterases (AXE). Eight AXE from different carbohydrate esterase (CE) families were tested on their activity against CA with a degree of substitution of 0.7 and 1.4. The classification of the AXEs into CE families according to their structure by hydrophobic cluster analysis followed clearly their activity against CA. Within the same CE family similar, and between the CE families different deacetylation behaviours could be observed. Furthermore, each esterase family showed a distinct regioselective mode of action. The CE 1 family enzymes regioselectively cleaved the substituents in C2- and C3-position, while CE 5 family enzymes only cleaved the acetyl groups in C2-position. CE 4 family enzymes seemed to interact only with the substituents in C3-position. Evidence was found that the deacetylation reaction of the CE 1 family enzymes proceeded faster in C2- than in C3-position of CA. The enzymes were able to cleave acetyl groups from fully substituted anhydroglucose units.

Comparison testing of methods for gel permeation chromatography of cellulose: coming closer to a standard protocol

A round robin on GPC of a wide range of different pulp samples was conducted among leading groups in cellulose analysis. The aim was to survey the status quo of the methods available to date. The pulp samples covered not only fully-bleached dissolving pulps but also bleached paper pulps and one unbleached sample. The methods applied were current state-of-the-art GPC with RI, MALLS, and viscosimetry detectors. Different dissolution protocols were compared as well. Following from the obtained results, more standardized protocols were proposed for approaches with different equipment (RI or MALLS/RI) and solvent systems (direct dissolution or derivatization). Major influencing factors, such as derivatization compared to direct solution, calibration versus light scattering and in-between lab variation, were discussed.

Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.

In Vitro Model Assemblies To Study the Impact of Lignin-Carbohydrate Interactions on the Enzymatic Conversion of Xylan

Endo-beta-1,4-xylanases (EC 3.2.1.8) are the main enzymes involved in the hydrolysis of xylans, the most abundant hemicelluloses in plant biomass. However, the development of efficient endoxylanases for use in biorefinery processes is currently hampered by insufficient knowledge regarding the impact of the cell wall network organization on the action of the enzyme at the supramolecular level. The action pattern of a GH11 endoxylanase from Thermobacillus xylanilyticus (Tx-xyl) was investigated by means of in vitro reconstituted model systems which can mimic certain cell wall structures. The action of Tx-xyl was evaluated on polymer assemblies displaying increasing complexity using delignified glucuronoarabinoxylan (GAX), then GAX-DHP model complexes obtained by oxidative polymerization of coniferyl alcohol into dehydrogenation polymers (DHP: lignin model compounds) in the presence of GAX. At a high concentration of GAX, interchain associations are formed leading to high molecular weight aggregates. These structures did not appear to affect the action of endoxylanase, which induces disaggregation of the self-aggregates along with polymer depolymerization. To mimic lignin-carbohydrate interactions, two different GAX-DHP nanocomposites were prepared and incubated with endoxylanase. In both cases, free GAX was hydrolyzed, while the GAX-DHP complexes appeared to be resistant. In the case of the noncovalently linked GAX-DHP(ZL) complexes, enzyme action favored a decrease in particle size, owing to the removal of their relatively exposed carbohydrate chains, whereas the complex supramolecular organization of the covalently linked GAX-DHP(ZT) complexes severely hampers the enzymes access to carbohydrate. Overall, these results establish the negative impact of DHP on the endoxylanase action and provide new knowledge regarding the limitations of the enzyme action in the lignocellulose bioconversion processes.