Lisbeth Garbrecht Thygesen

Cell-wall structural changes in wheat straw pretreated for bioethanol production.

BackgroundPretreatment is an essential step in the enzymatic hydrolysis of biomass and subsequent production of bioethanol. Recent results indicate that only a mild pretreatment is necessary in an industrial, economically feasible system. The Integrated Biomass Utilisation System hydrothermal pretreatment process has previously been shown to be effective in preparing wheat straw for these processes without the application of additional chemicals. In the current work, the effect of the pretreatment on the straw cell-wall matrix and its components are characterised microscopically (atomic force microscopy and scanning electron microscopy) and spectroscopically (attenuated total reflectance Fourier transform infrared spectroscopy) in order to understand this increase in digestibility.ResultsThe hydrothermal pretreatment does not degrade the fibrillar structure of cellulose but causes profound lignin re-localisation. Results from the current work indicate that wax has been removed and hemicellulose has been partially removed. Similar changes were found in wheat straw pretreated by steam explosion.ConclusionResults indicate that hydrothermal pretreatment increases the digestibility by increasing the accessibility of the cellulose through a re-localisation of lignin and a partial removal of hemicellulose, rather than by disruption of the cell wall.

A critical discussion of the physics of wood–water interactions

This paper reviews recent findings on wood–water interaction and puts them into context of established knowledge in the field. Several new findings challenge prevalent theories and are critically discussed in an attempt to advance current knowledge and highlight gaps. The focus of this review is put on water in the broadest concept of wood products, that is, the living tree is not considered. Moreover, the review covers the basic wood–water relation, states and transitions. Secondary effects such as the ability of water to alter physical properties of wood are only discussed in cases where there is an influence on state and/or transition.

Vibrational microspectroscopy of food. Raman vs. FT-IR

FT-IR and Raman spectroscopy are complementary techniques for the study of molecular vibrations and structure. The combination with a microscope results in an analytical method that allows spatially resolved investigation of the chemical composition of heterogeneous foods and food ingredients. The high spatial resolution makes it possible to study areas down to approximately 10×10 μm with FT-IR microspectroscopy and approximately 1×1 μm with Raman microspectroscopy. This presentation highlights the advantages and disadvantages of the two microspectroscopic techniques when applied to different heterogeneous food systems. FT-IR and Raman microspectroscopy were applied to a number of different problems related to food analysis: (1) in situ determination of starch and pectin in the potato cell, (2) in situ determination of the distribution of amygdalin in bitter almonds, (3) the composition of blisters found on the surface of bread, (4) the microstructure of high-lysine barley and (5) the composition of white spots in the shell of frozen shrimps.

Cellulase inhibition by high concentrations of monosaccharides.

Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The β-glucosidases were shown to be less sensitive to high monosaccharide concentrations except glucose. Protein adsorption studies showed that this inhibition effect was most likely due to catalytic, and not binding, inhibition of the cellulases.

Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass.

Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free‐fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (Aw) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low‐field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels.

Equilibrium moisture content (EMC) in Norway spruce during the first and second desorptions

Abstract It is a commonly accepted notion that the equilibrium moisture content (EMC) of wood at a given relative humidity (RH) is highest during initial desorption of green wood due to an irreversible loss of hygroscopicity during the 1st desorption. The basis for this notion is investigated by assessing how drying and saturation procedures influence the differences between the 1st and the 2nd desorption curves for Norway spruce (Picea abies (L.) Karst.) sapwood. The study establishes 1st and 2nd desorption isotherms for a variety of initial conditions and it covers the RH range from 60.1% to 99.9%. The state of the water is not affected by oven-drying and rewetting as demonstrated by time domain low field NMR relaxometry. The results challenge the conclusions of earlier studies and indicate that in these studies the 2nd desorption was initiated at much too low EMC and therefore fails to describe a boundary desorption isotherm. Instead, it becomes an intermediate desorption isotherm starting at the adsorption boundary curve and crossing over to eventually meet the desorption boundary curve. The results also show that vacuum drying at room temperature only gives a modest loss of hygroscopicity compared to the green state. Conversely, oven-drying at 103°C results in a more significant loss of hygroscopicity, except for RH above 96% where an increase in EMC surprisingly is seen.

A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

BackgroundEnzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.ResultsWe have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained.ConclusionsWe show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.

The effects of growth and storage conditions on dislocations in hemp fibres

Natural fibres contain structural distortions known as dislocations. Dislocations have been found to negatively affect the properties of plant fibre-based materials. The present study concerned the effect of the growth conditions of hemp plants (Cannabis sativa L.) on the amount of dislocations within the fibres retrieved from the stems. Dislocations were found in fibres from plants grown under absolutely wind-free conditions, but both windy and dry conditions introduced significantly more dislocations. Furthermore their maximum size was larger and they were situated closer together within the fibres. The average ultimate tensile stress of fibre bundles from the windy and the dry growth regimes was lower than that of fibre bundles from plants grown under wind-free conditions. In a separate study it was found that plant stems that were dried after harvest and remoistened prior to analysis contained a lower amount of dislocations than plants analysed directly after harvest.

Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose saccharification

BackgroundStudies in bioconversions have continuously sought the development of processing strategies to overcome the “close physical association” between plant cell wall polymers thought to significantly contribute to biomass recalcitrance [Adv Space Res 18:251–265, 1996],[ Science 315:804–807, 2007]. To a lesser extent, studies have sought to understand biophysical factors responsible for the resistance of lignocelluloses to enzymatic degradation. Provided here are data supporting our hypothesis that the inhibitory potential of different cell wall polymers towards enzymatic cellulose hydrolysis is related to how much these polymers constrain the water surrounding them. We believe the entropy-reducing constraint imparted to polymer associated water plays a negative role by increasing the probability of detrimental interactions such as junction zone formation and the non-productive binding of enzymes.ResultsSelected commercial lignocellulose-derived polymers, including hemicelluloses, pectins, and lignin, showed varied potential to inhibit 24-h cellulose conversion by a mix of purified cellobiohydrolase I and β-glucosidase. At low dry matter loadings (0.5% w/w), insoluble hemicelluloses were most inhibitory (reducing conversion relative to cellulose-only controls by about 80%) followed by soluble xyloglucan and wheat arabinoxylan (reductions of about 70% and 55%, respectively), while the lignin and pectins tested were the least inhibitory (approximately 20% reduction). Low field nuclear magnetic resonance (LF-NMR) relaxometry used to observe water-related proton relaxation in saturated polymer suspensions (10% dry solids, w/w) showed spin-spin, T2, relaxation time curves generally approached zero faster for the most inhibitory polymer preparations. The manner of this decline varied between polymers, indicating different biophysical aspects may differentially contribute to overall water constraint in each case. To better compare the LF-NMR data to inhibitory potential, T2 values from monocomponent exponential fits of relaxation curves were used as a measure of overall water constraint. These values generally correlated faster relaxation times (greater water constraint) with greater inhibition of the model cellulase system by the polymers.ConclusionsThe presented correlation of cellulase inhibition and proton relaxation data provides support for our water constraint-biomass recalcitrance hypothesis. Deeper investigation into polymer-cellulose-cellulase interactions should help elucidate the types of interactions that may be propagating this correlation. If these observations can be verified to be more than correlative, the hypothesis and data presented suggest that a focus on water-polymer interactions and ways to alter them may help resolve key biological lignocellulose processing bottlenecks.

Cellulose is not just cellulose: a review of dislocations as reactive sites in the enzymatic hydrolysis of cellulose microfibrils

Most secondary plant cell walls contain irregular regions known as dislocations or slip planes. Under industrial biorefining conditions dislocations have recently been shown to play a key role during the initial phase of the enzymatic hydrolysis of cellulose in plant cell walls. In this review we chart previous publications that have discussed the structure of dislocations and their susceptibility to hydrolysis. The supramolecular structure of cellulose in dislocations is still unknown. However, it has been shown that cellulose microfibrils continue through dislocations, i.e. dislocations are not regions where free cellulose ends are more abundant than in the bulk cell wall. In more severe cases cracks between fibrils form at dislocations and it is possible that the increased accessibility that these cracks give is the reason why hydrolysis of cellulose starts at these locations. If acid or enzymatic hydrolysis of plant cell walls is carried out simultaneously with the application of shear stress, plant cells such as fibers or tracheids break at their dislocations. At present it is not known whether specific carbohydrate binding modules (CBMs) and/or cellulases preferentially access cellulose at dislocations. From the few studies published so far it seems that no special type of CBM is involved. In one case an endoglucanase was found to preferably bind to dislocations.