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A combination of thermochemical pretreatment and biological digestion technologies is usually required to overcome lignocellulosic recalcitrance and accomplish effective biomass deconstruction. This study is aimed at understanding switchgrass breakdown by hydrothermal, dilute acid, dilute alkali, and co-solvent enhanced lignocellulosic fractionation (CELF) pretreatments followed by application of traditional fungal enzymatic hydrolysis (EH) and Clostridium thermocellum consolidated bioprocessing (CBP) to the resulting solids. Unpretreated and pretreated switchgrass and their EH and CBP residues were characterized by a suite of analytical techniques to understand structural changes that occurred during deconstruction. CELF pretreated solids showed the highest accessibility and digestibility by both EH and CBP followed by dilute alkali and then dilute acid/hydrothermal pretreated solids. Lignin removal from biomass had a more positive impact on substrate accessibility and digestibility than did xylan removal, while xyloglucan removal by pretreatment appeared essential for cellulose digestion by fungal enzymes. The extent of CBP digestion of cellulose and non-cellulosic glycans was larger than that by EH. Unlike dilute alkali pretreatment, cellulose crystallinity increased for acid-based pretreatments in the following order: hydrothermal, dilute acid, and CELF. Acid-based pretreatments also substantially reduced cellulose degree of polymerization. All thermochemical and biological digestion approaches increased syringyl to guaiacyl lignin (S/G) ratio and reduced β-O-4 lignin interunit linkage and hydroxycinnamates content from levels in unpretreated switchgrass. The substantial increase in S/G ratio after hydrothermal and dilute alkali preatreatments suggested that high temperatures or alkali removed a large portion of G lignin from switchgrass.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

Background The rumen microbial community is considered the most efficient anaerobic digestive ecosystem known, yet less than half of the energy in low quality forages is actually metabolized. There is a knowledge gap regarding the specific factors that impede the ruminal digestion of plant cell walls or if rumen microbiota have the functional potential and activities to overcome these constraints. To address these issues, innovative experimental methods may provide a high-resolution understanding of the cell wall chemistries and higher-order structures that are resistant to microbial digestion and how they interact with the functional activities of the rumen microbial community. Results With this goal, we characterized the total tract indigestible residue (TTIR) from cattle fed a high-forage diet containing low-quality straw using two comparative glycomic approaches: ELISA-based glycome profiling and glycosidic linkage analysis. Using these techniques, we successfully detected numerous and diverse cell wall glycan epitopes in barley straw and TTIR and determined their relative abundance pre- and post-intestinal digestion. Of these, xyloglucans and heteroxylans were the most recalcitrant to digestion. Linkage analysis identified indigestible linkages consistent with the polysaccharide epitopes identified by ELISA-based glycome analysis. To determine if residual plant polysaccharides within TTIR could be metabolised, rumen microbiota from cannulated cattle fed barley straw were incubated with barley straw and TTIR in in vitro batch cultures. Transcript coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences revealed encoded enzymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. Conclusion This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can inform next-generation solutions to improve the growth of ruminants fed low quality forages and enhance the use of crop residues as a feedstock.

Background Downregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized. Results Double fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis. Conclusion Our observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects. Electronic supplementary material The online version of this article (10.1186/s13068-019-1446-3) contains supplementary material, which is available to authorized users.

Additional file 2: Fig. S1. The phenotype of 9-day-old seedlings of WT, fpgs1, ccoaomt1 and fpgs1ccoaomt1 plants. Similar to fpgs1 single mutants, fpgs1ccoaomt1double mutants had short roots during early development.

Lignocellulosic fibers are plant-based bio-fibers that are sourced from terrestrial and non-terrestrial plants that also include agricultural by-products. In textile industry, being a renewable and sustainable resource, these fibers have attained market potential with their special qualities including biodegradability, applicability in green chemistry, ecofriendly properties and cost effectiveness in processing. Lignocellulosic fibers mainly comprise plant cell walls that are essentially composed of cellulose, lignin, pectin, hemicelluloses and waxes. Numerous studies have been conducted to explain the commercial performance, mechanical properties and sustainability of these lignocellulosic fibers. Although several work have been conducted on the cell wall compositional aspects of these fibers, majority of the studies have been focused on understanding the cellulose and lignin constituents. Hence, there is an increasing need for more studies to be conducted on non-cellulosic cell wall components in these fibers. In this study, we performed a novel immunological approach namely glycome profiling on two commercially important lignocellulosic fibers from aquatic and terrestrial plants namely duckweed and hemp, respectively. Our studies demonstrated differences in the extractability of cell wall glycans from biomass materials from these plants. Duckweed biomass had significantly higher abundance of extractable pectins in their walls. However, cell walls from biomass raw materials of terrestrial plant, hemp had lesser amounts of pectin with significantly higher amounts of xylans that are easily extractable. Duckweed fibers had significantly higher proportion of lignin-associated cell wall glycans in comparison to that of hemp. These results demonstrate that lignocellulosic fiber raw materials from varied plant sources are different in non-cellulosic cell wall glycan compositions and comprehending these variations could potentially be instrumental in commercial processing of these fibers.