Enzymatic hydrolysis of the gelatinous layer in tension wood of Salix varieties as a measure of accessible cellulose for biofuels

Abstract

Background Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing d-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free d-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). Results Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of d-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free d-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. Conclusions The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available d-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with d-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with d-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced d-glucose release in Salix varieties.

Evaluation of Wood Quality Traits in Salix viminalis Useful for Biofuels: Characterization and Method Development

“…Light microscopy observations confirmed the presence of typical tension wood fibers (G-fibers) with pronounced gelatinous layers (Figure 4A-D). As reported previously, the G-layers appeared weakly attached to the outer secondary cell wall layer in tension fibers (i.e., S2 layer) and, after sectioning, were frequently detached (Figure 4D arrows; [35,41]). Observations of tension wood bands showed variability in the thickness of the G-layer depending on presence in earlywood (EW)/latewood (LW) and between clone samples (Figure 4E,F).…”

“…Sections were double-stained with 1% w/v chlorazol black E and 0.1% w/v safranin to visualize the presence of tension wood (TW) [34]. Chlorazol black E strongly stains the gelatinous layer (G-layer) of TW fibers black with our previous studies showing the combination of safranin and chlorazol black E giving the best staining for image analysis [35]. Entire stem cross-sections were stained and scanned using an Epson Perfection Pro 750 film scanner with a pixel resolution of 2400 dpi and areas of TW marked.…”

“…All stains were purchased from Sigma-Aldrich (St. Louis, MO, USA). Additional sections were stained with 1% w/v astra blue [35] or processed for scanning electron microscopy (SEM; [38]) for morphological observations and measurements on G-layer thickness.…”

“…2-4 mm in diameter) was removed. Samples were delignified in a 1:1 mixture of 100% acetic acid and H 2 O 2 at 60 • C overnight [35,39]. Following delignification, samples were washed (5 × distilled water) and macerated by agitation for 2 h, thus forming a fiber suspension for morphological analysis of cellular elements.…”

“…For comparison, in one example, the vessel frequency determined using the OFA was compared with direct quantification (i.e., counting) of vessel frequency in semi-thin stem sections of S. viminalis clone 81041 TW and OW using scanning electron microscopy (SEM). S. viminalis sections were cut and prepared for SEM as previously described [35].…”