Biological Lignocellulose Solubilization: Comparative Evaluation of Biocatalysts and Enhancement Via Cotreatment

Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions

The Effects of High Concentrations of Ionic Liquid on GB1 Protein Structure and Dynamics Probed by High-Resolution Magic-Angle-Spinning NMR Spectroscopy

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentrations of ionic liquids, has been challenging. In the present work the 13C, 15N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid–protein interactions. Effect of an ionic liquid (1-butyl-3- methylimidazolium bromide, [C4-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6–3.5 M, which corresponds to 10–60% v/v). Interactions between GB1 and [C4-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C4-mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of molecular mechanisms of ionic liquid–protein interactions

Compensatory guaiacyl lignin biosynthesis at the expense of syringyl lignin in 4CL1-knockout poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production

Background Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold. Results We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance. Conclusions Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production. Keywords: Switchgrass; Bioenergy; Biofuel; Feedstock; Cellulosic ethanol; PvMYB4; Transcription factor; Cell wall; Recalcitrance; Lignin; Hemicellulose; Pecti

Solid-State Selective ¹³C Excitation and Spin Diffusion NMR To Resolve Spatial Dimensions in Plant Cell Walls

The average spatial dimensions between major biopolymers within the plant cell wall can be resolved using a solid-state NMR technique referred to as a ¹³C cross-polarization (CP) SELDOM (<i>sel</i>ectively by <i>d</i>estruction <i>o</i>f <i>m</i>agnetization) with a mixing time delay for spin diffusion. Selective excitation of specific aromatic lignin carbons indicates that lignin is in close proximity to hemicellulose followed by amorphous and finally crystalline cellulose. ¹³C spin diffusion time constants (<i>T</i>_SD) were extracted using a two-site spin diffusion theory developed for ¹³C nuclei under magic angle spinning (MAS) conditions. These time constants were then used to calculate an average lower-limit spin diffusion length between chemical groups within the plant cell wall. The results on untreated ¹³C enriched corn stover stem reveal that the lignin carbons are, on average, located at distances ∼0.7–2.0 nm from the carbons in hemicellulose and cellulose, whereas the pretreated material had larger separations

Hydrotreating the Organic Fraction of Biomass Pyrolysis Oil to a Refinery Intermediate

The effect of the catalyst type, hydroprocessing conditions, and feed preparation on the mild hydrotreating of biomass pyrolytic lignin was examined. Pyrolytic lignin oils were produced by water separation at 1:1 and 3:1 water/oil mass ratios. Hydrotreating was performed in a semi-batch reactor at three severities using sulfided NiMo/Al₂O₃, Pd/C, and Pt/C catalysts. Over half of the carbon in the pyrolytic lignin could be converted to a low-oxygen (<5%), low-acid, volatile, hydrocarbon-miscible liquid product. This was achieved with all three catalysts at the most severe condition (400 °C and 2450 psig) and with Pt/C at somewhat less severe conditions. Nuclear magnetic resonance (NMR) analyses indicated that the remaining oxygen is largely phenolic in character. Hydrotreating of the organic fraction (pyrolytic lignin) gave oil with better properties, lower O and lower acidity, than hydrotreating of the whole oil at equivalent conditions