Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. ‘‘Alamo’’ with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the TDNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol- HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin

Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

Genetic modification of herbaceous plant cell walls to increase biofuels yields is a primary bioenergy research goal. Using two switchgrass populations developed by divergent breeding for ruminant digestibility, the contributions of several wall-related factors to ethanol yields was evaluated. Field grown low lignin plants significantly out yielded high lignin plants for conversion to ethanol by 39.1% and extraction of xylans by 12%. However, across all plants analyzed, greater than 50% of the variation in ethanol yields was attributable to changes in tissue and cell wall architecture, and responses of stem biomass to diluteacid pretreatment. Although lignin levels were lower in the most efficiently converted genotypes, no apparent correlation were seen in the lignin monomer G/S ratios. Plants with higher ethanol yields were associated with an apparent decrease in the lignification of the cortical sclerenchyma, and a marked decrease in the granularity of the cell walls following dilute-acid pretreatment

Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

Genetic modification of herbaceous plant cell walls to increase biofuels yields is a primary bioenergy research goal. Using two switchgrass populations developed by divergent breeding for ruminant digestibility, the contributions of several wall-related factors to ethanol yields was evaluated. Field grown low lignin plants significantly out yielded high lignin plants for conversion to ethanol by 39.1% and extraction of xylans by 12%. However, across all plants analyzed, greater than 50% of the variation in ethanol yields was attributable to changes in tissue and cell wall architecture, and responses of stem biomass to diluteacid pretreatment. Although lignin levels were lower in the most efficiently converted genotypes, no apparent correlation were seen in the lignin monomer G/S ratios. Plants with higher ethanol yields were associated with an apparent decrease in the lignification of the cortical sclerenchyma, and a marked decrease in the granularity of the cell walls following dilute-acid pretreatment

Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Overexpression of \u3ci\u3eSbMyb60\u3c/i\u3e impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in \u3ci\u3eSorghum bicolor\u3c/i\u3e

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses