Here we report the production of highly thermal stable and functional cellulose nanocrystals (CNC) and nanofibrils (CNF) by hydrolysis using concentrated organic acids. Due to their low water solubility, these solid organic acids can be easily recovered after hydrolysis reactions through crystallization at a lower or ambient temperature. When dicarboxylic acids were used, the resultant CNC surface contained carboxylic acid groups which facilitate functionalization and dispersion in aqueous processing. The carboxylic acid group content was 0.1–0.4 mmol g⁻¹ for CNC produced from a bleached eucalyptus kraft pulp (BEP) using oxalic acid at concentrations of 50–70 wt%. The onset thermal degradation temperature for the CNC was increased to 322 °C from 274 °C for the feed BEP fibers, compared with 218 °C for CNC produced from the same feed fibers using conventional concentrated sulfuric acid hydrolysis. The low strength (high pK_a) of organic acids also resulted in CNC with longer lengths of approximately 275–380 nm and higher crystallinity than those produced using mineral acids. Fibrous cellulosic solid residue (FCSR) collected from acid hydrolysis was an excellent feedstock for producing CNF through subsequent mechanical fibrillation with low energy input. The ability to recover organic acids using a conventional and commercially proven crystallization method makes these organic acids uniquely suitable for sustainable and green production of cellulose nanomaterials. The resultant CNC and CNF with high thermal stability and a large aspect ratio are excellent for bio-composite applications.

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

We studied xylem anatomy and hydraulic architecture in 14 transgenic insertion events and a control line of hybrid poplar (Populus spp.) that varied in lignin content. Transgenic events had different levels of down‐regulation of two genes encoding 4‐coumarate:coenzyme A ligase (4CL). Two‐year‐old trees were characterized after growing either as free‐standing trees in the field or as supported by stakes in a greenhouse. In free‐standing trees, a 20 to 40% reduction in lignin content was associated with increased xylem vulnerability to embolism, shoot dieback and mortality. In staked trees, the decreased biomechanical demands on the xylem was associated with increases in the leaf area to sapwood area ratio and wood specific conductivity (k_s), and with decreased leaf‐specific conductivity (k_l). These shifts in hydraulic architecture suggest that the bending stresses perceived during growth can affect traits important for xylem water transport. Severe 4CL‐downregulation resulted in the patchy formation of discoloured, brown wood with irregular vessels in which water transport was strongly impeded. These severely 4CL‐downregulated trees had significantly lower growth efficiency (biomass/leaf area). These results underscore the necessity of adequate lignification for mechanical support of the stem, water transport, tree growth and survival.

Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.