The contribution of G-layer glucose in Salix clones for biofuels: comparative enzymatic and HPLC analysis of stem cross sections
Authors: Gao Jie , Jebrane Mohamed , Terziev Nasko , Daniel Geoffrey 
Published: March 2022

Abstract
Background
Interest on the use of short rotation willow as a lignocellulose resource for liquid transport fuels has increased greatly over the last 10 years. Investigations have shown the advantages and potential of using Salix spp. for such fuels but have also emphasized the wide variations existing in the compositional structure between different species and genotypes in addition to their effects on overall yield. The present work studied the importance of tension wood (TW) as a readily available source of glucose in 2-year-old stems of four Salix clones (Tora, Björn, Jorr, Loden). Studies involved application of a novel approach whereby TW-glucose and residual sugars and lignin were quantified using stem cross sections with results correlated with HPLC analyses of milled wood. Compositional analyses were made for four points along stems and glucose derived from enzyme saccharification of TW gelatinous (G) layers (G-glucose), structural cell wall glucose (CW-glucose) remaining after saccharification and total glucose (T-glucose) determined both theoretically and from HPLC analyses. Comparisons were also made between presence of other characteristic sugars as well as acid-soluble and -insoluble lignin.

Results
Preliminary studies showed good agreement between using stem serial sections and milled powder from Salix stems for determining total sugar and lignin. Therefore, sections were used throughout the work. HPLC determination of T-glucose in Salix clones varied between 47.1 and 52.8%, showing a trend for higher T-glucose with increasing height (Björn, Tora and Jorr). Using histochemical/microscopy and image analysis, Tora (24.2%) and Björn (28.2%) showed greater volumes of % TW than Jorr (15.5%) and Loden (14.0%). Total G-glucose with enzyme saccharification of TW G-layers varied between 3.7 and 14.7% increasing as the total TW volume increased. CW-glucose measured after enzyme saccharification showed mean values of 41.9–49.1%. Total lignin between and within clones showed small differences with mean variations of 22.4–22.8% before and 22.4–24.3% after enzyme saccharification. Calculated theoretical and quantified values for CW-glucose at different heights for clones were similar with strong correlation: T-glucose = G-glucose + CW-glucose. Pearson’s correlation displayed a strong and positive correlation between T-glucose and G-glucose, % TW and stem height, and between G-glucose with % TW and stem height.

Conclusions
The use of stem cross sections to estimate TW together with enzyme saccharification represents a viable approach for determining freely available G-glucose from TW allowing comparisons between Salix clones. Using stem sections provides for discrete morphological/compositional tissue comparisons between clones with results consistent with traditional wet chemical analysis approaches where entire stems are milled and analyzed. The four clones showed variable TW and presence of total % G-glucose in the order Björn > Tora > Jorr > Loden. Calculated in terms of 1 m3, Salix stems Tora and Björn would contain ca. 0.24 and 0.28 m3 of tension wood representing a significant amount of freely available glucose.

Graphical Abstract