Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.

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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 3 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.Composed of diverse layers of cellulose microfibrils and amorphous hemicelluloses within a matrix of pectins, proteins, and lignin, the secondary cell walls of plants are diverse in their morphology, chemistry, and physiological functions. Lignification is of particular interest, as it exhibits highly predictable temporal and spatial patterning and is the last major step in the structural reinforcement of cell walls before the protoplast is dissolved (Donaldson, 2001

The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

BackgroundThe inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner.ResultsGAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines.ConclusionsThe results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.

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Nature has evolved multiple enzymatic strategies for the degradation of plant cell wall polysaccharides, which are central to carbon flux in the biosphere and an integral part of renewable biofuels production.Many biomass-degrading organisms secrete synergistic cocktails of individual enzymes with one or several catalytic domains per enzyme, whereas a few bacteria synthesize large multi-enzyme complexes, termed cellulosomes, which contain multiple catalytic units per complex. Both enzyme systems employ similar catalytic chemistries; however, the physical mechanisms by which these enzyme systems degrade polysaccharides are still unclear. Here we examine a prominent example of each type, namely a freeenzyme cocktail expressed by the fungus Hypocrea jecorina and a cellulosome preparation secreted from the anaerobic bacterium Clostridium thermocellum. We observe striking differences in cellulose saccharification exhibited by these systems at the same protein loading. Free enzymes are more active on pretreated biomass and in contrast cellulosomes are much more active on purified cellulose. When combined, these systems display dramatic synergistic enzyme activity on cellulose. To gain further insights, we imaged free enzyme-and cellulosome-digested cellulose and biomass by transmission electron microscopy, which revealed evidence for different mechanisms of cellulose deconstruction by free enzymes and cellulosomes. Specifically, the free enzymes employ an ablative, fibril-sharpening mechanism, whereas cellulosomes physically separate individual cellulose microfibrils from larger particles resulting in enhanced access to cellulose surfaces. Interestingly, when the two enzyme systems are combined, we observe changes to the substrate that suggests mechanisms of synergistic deconstruction. Insight into the different mechanisms underlying these two polysaccharide deconstruction paradigms will eventually enable new strategies for enzyme engineering to overcome biomass recalcitrance. Broader contextIndustrial conversion of plant biomass into transportation fuels will likely be a vital component of the global renewable energy portfolio to reduce both greenhouse gas emissions and fossil fuel utilization for mankind's energy needs. However, plants have evolved considerable defense mechanisms against deconstruction of their cell wall polysaccharides into sugars, and as such, depolymerization to sugars for subsequent biological or catalytic conversion to fuels remains a signicant technical challenge with major cost implications in biomass conversion. In nature, many microorganisms evolved a strategy wherein plant cell wall-active enzymes are secreted as a cocktail of individual enzymes that work synergistically to depolymerize biomass, referred to as a "free enzyme" paradigm. Some ruminal microorganisms conversely evolved a strategy wherein their plant cell wall-active enzymes are tethered to large scaffolds, which are linked to the cells for sugar production in close proximity for uptake, termed the "cellulosome". Here, we mecha...