The study of biomass deconstruction by enzymatic hydrolysis has hitherto not focussed on the importance of supramolecular structures of cellulose. In lignocellulose fibres, regions with a different organisation of the microfibrils are present. These regions are called dislocations or slip planes and they are known to be more susceptible to various forms of degradation such as acid hydrolysis. Traditionally the cellulose within these regions has been assumed to be amorphous, but in this study it is shown by use of polarized light microscopy that dislocations are birefringent. This indicates that they have a crystalline organisation. Dislocations may be entry points for endoglucanases. Using a fluorescent labelled endoglucanase combined with confocal fluorescence microscopy, it is shown that the enzyme selectively binds to dislocations during the initial phase of the hydrolysis. Using a commercial cellulase mixture on hydrothermally treated wheat straw, it was found that the fibres were cut into segments corresponding to the sections between the dislocations initially present, as has previously been observed for acid hydrolysis of softwood pulps. The results indicate that dislocations are important during the initial part of enzymatic hydrolysis of cellulose. The implications of this phenomenon have not yet been recognized or explored within cellulosic biofuels.

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.

BackgroundWheat straw used for bioethanol production varies in enzymatic digestibility according to chemical structure and composition of cell walls and tissues. In this work, the two biologically different wheat straw organs, leaves and stems, are described together with the effects of hydrothermal pretreatment on chemical composition, tissue structure, enzyme adhesion and digestion. To highlight the importance of inherent cell wall characteristics and the diverse effects of mechanical disruption and biochemical degradation, separate leaves and stems were pretreated on lab-scale and their tissue structures maintained mostly intact for image analysis. Finally, samples were enzymatically hydrolysed to correlate digestibility to chemical composition, removal of polymers, tissue composition and disruption, particle size and enzyme adhesion as a result of pretreatment and wax removal. For comparison, industrially pretreated wheat straw from Inbicon A/S was included in all the experiments.ResultsWithin the same range of pretreatment severities, industrial pretreatment resulted in most hemicellulose and epicuticular wax/cutin removal compared to lab-scale pretreated leaves and stems but also in most re-deposition of lignin on the surface. Tissues were furthermore degraded from tissues into individual cells while lab-scale pretreated samples were structurally almost intact. In both raw leaves and stems, endoglucanase and exoglucanase adhered most to parenchyma cells; after pretreatment, to epidermal cells in all the samples. Despite heavy tissue disruption, industrially pretreated samples were not as susceptible to enzymatic digestion as lab-scale pretreated leaves while lab-scale pretreated stems were the least digestible.ConclusionsDespite preferential enzyme adhesion to epidermal cells after hydrothermal pretreatment, our results suggest that the single most important factor determining wheat straw digestibility is the fraction of parenchyma cells rather than effective tissue disruption.

The production of acid phosphatases (E.C.3.1.3.2, ACPs) by Aspergillus niger N402A is regulated by specific growth rate, as well as phosphate availability and pH, as demonstrated by studies in continuous flow culture. Specific ACP activity was highest when A. niger was grown at pH 6.3 (64+/-8 U g(-1)) or pH 2.8 (99+/-11 U g(-1)), at a dilution rate of 0.07 h(-1) and phosphate concentrations below 0.46 mM. ACP production was growth correlated for specific growth rates between 0.07 and 0.13 h(-1). Four different ACPs, including two phytases, were produced by A. niger N402A. The ACP and the phytase with maximal activities at pH 5.5 were differentially expressed at different culture pH values, with greater production at low pH.

Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.