Management controls the net greenhouse gas outcomes of growing bioenergy feedstocks on marginally productive croplands.

Bio-based energy is key to developing a globally sustainable low-carbon economy. Lignocellulosic feedstock production on marginally productive croplands is expected to provide substantial climate mitigation benefits, but long-term field research comparing greenhouse gas (GHG) outcomes during the production of annual versus perennial crop-based feedstocks is lacking. Here, we show that long-term (16 years) switchgrass (Panicum virgatum L.) systems mitigate GHG emissions during the feedstock production phase compared to GHG-neutral continuous corn (Zea mays L.) under conservation management on marginally productive cropland. Increased soil organic carbon was the major GHG sink in all feedstock systems, but net agronomic GHG outcomes hinged on soil nitrous oxide emissions controlled by nitrogen (N) fertilizer rate. This long-term field study is the first to demonstrate that annual crop and perennial grass systems respectively maintain or mitigate atmospheric GHG contributions during the agronomic phase of bioenergy production, providing flexibility for land-use decisions on marginally productive croplands.

Selection Signatures in Four Lignin Genes from Switchgrass Populations Divergently Selected for In Vitro Dry Matter Digestibility.

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four loci in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. This study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.

Accuracy of Genomic Prediction in Switchgrass (Panicum virgatum L.) Improved by Accounting for Linkage Disequilibrium.

Switchgrass is a relatively high-yielding and environmentally sustainable biomass crop, but further genetic gains in biomass yield must be achieved to make it an economically viable bioenergy feedstock. Genomic selection (GS) is an attractive technology to generate rapid genetic gains in switchgrass, and meet the goals of a substantial displacement of petroleum use with biofuels in the near future. In this study, we empirically assessed prediction procedures for genomic selection in two different populations, consisting of 137 and 110 half-sib families of switchgrass, tested in two locations in the United States for three agronomic traits: dry matter yield, plant height, and heading date. Marker data were produced for the families' parents by exome capture sequencing, generating up to 141,030 polymorphic markers with available genomic-location and annotation information. We evaluated prediction procedures that varied not only by learning schemes and prediction models, but also by the way the data were preprocessed to account for redundancy in marker information. More complex genomic prediction procedures were generally not significantly more accurate than the simplest procedure, likely due to limited population sizes. Nevertheless, a highly significant gain in prediction accuracy was achieved by transforming the marker data through a marker correlation matrix. Our results suggest that marker-data transformations and, more generally, the account of linkage disequilibrium among markers, offer valuable opportunities for improving prediction procedures in GS. Some of the achieved prediction accuracies should motivate implementation of GS in switchgrass breeding programs.

Multi-Year Pathogen Survey of Biofuel Switchgrass Breeding Plots Reveals High Prevalence of Infections by Panicum mosaic virus and Its Satellite Virus.

Switchgrass (Panicum virgatum) cultivars are currently under development as lignocellulosic feedstock. Here we present a survey of three established switchgrass experimental nurseries in Nebraska in which we identified Panicum mosaic virus (PMV) as the most prevalent virus. In 2012, 72% of 139 symptomatic plants tested positive for PMV. Of the PMV-positive samples, 19% were coinfected with its satellite virus (SPMV). Less than 14% of all sampled plants in 2012 were positive for four additional viruses known to infect switchgrass. In 2013, randomized sampling of switchgrass individuals from the same 2012 breeding plots revealed that infection by PMV or PMV+SPMV was both more prevalent and associated with more severe symptoms in the cultivar Summer, and experimental lines with Summer parentage, than populations derived from the cultivar Kanlow. A 3-year analysis, from 2012 to 2014, showed that previously uninfected switchgrass plants acquire PMV or PMV+SPMV between harvest cycles. In contrast, some plants apparently did not maintain PMV infections at detectable levels from year-to-year. These findings suggest that PMV and SPMV should be considered important pathogens of switchgrass and serious potential threats to biofuel crop production efficiency.

Contrasting metabolism in perenniating structures of upland and lowland switchgrass plants late in the growing season.

BACKGROUND: Switchgrass (Panicum virgatum L.) is being developed as a bioenergy crop for many temperate regions of the world. One way to increase biomass yields is to move southern adapted lowland cultivars to more northern latitudes. However, many southerly adapted switchgrass germplasm can suffer significant winter kill in northerly climes. MATERIALS AND METHODS: Here, we have applied next-generation sequencing in combination with biochemical analyses to query the metabolism of crowns and rhizomes obtained from two contrasting switchgrass cultivars. Crowns and rhizomes from field-grown lowland (cv Kanlow) and upland (cv Summer) switchgrass cultivars were collected from three randomly selected post-flowering plants. Summer plants were senescing, whereas Kanlow plants were not at this harvest date. RESULTS: Principal component analysis (PCA) differentiated between both the Summer and Kanlow transcriptomes and metabolomes. Significant differences in transcript abundances were detected for 8,050 genes, including transcription factors such as WRKYs and those associated with phenylpropanoid biosynthesis. Gene-set enrichment analyses showed that a number of pathways were differentially up-regulated in the two populations. For both populations, protein levels and enzyme activities agreed well with transcript abundances for genes involved in the phenylpropanoid pathway that were up-regulated in Kanlow crowns and rhizomes. The combination of these datasets suggests that dormancy-related mechanisms had been triggered in the crowns and rhizomes of the Summer plants, whereas the crowns and rhizomes of Kanlow plants had yet to enter dormancy. CONCLUSIONS: Delayed establishment of dormancy at more northerly latitudes could be one factor that reduces winter-survival in the high-yielding Kanlow plants. Understanding the cellular signatures that accompany the transition to dormancy can be used in the future to select plants with improved winter hardiness.

Energy potential and greenhouse gas emissions from bioenergy cropping systems on marginally productive cropland.

Low-carbon biofuel sources are being developed and evaluated in the United States and Europe to partially offset petroleum transport fuels. Current and potential biofuel production systems were evaluated from a long-term continuous no-tillage corn (Zea mays L.) and switchgrass (Panicum virgatum L.) field trial under differing harvest strategies and nitrogen (N) fertilizer intensities to determine overall environmental sustainability. Corn and switchgrass grown for bioenergy resulted in near-term net greenhouse gas (GHG) reductions of -29 to -396 grams of CO2 equivalent emissions per megajoule of ethanol per year as a result of direct soil carbon sequestration and from the adoption of integrated biofuel conversion pathways. Management practices in switchgrass and corn resulted in large variation in petroleum offset potential. Switchgrass, using best management practices produced 3919±117 liters of ethanol per hectare and had 74±2.2 gigajoules of petroleum offsets per hectare which was similar to intensified corn systems (grain and 50% residue harvest under optimal N rates). Co-locating and integrating cellulosic biorefineries with existing dry mill corn grain ethanol facilities improved net energy yields (GJ ha-1) of corn grain ethanol by >70%. A multi-feedstock, landscape approach coupled with an integrated biorefinery would be a viable option to meet growing renewable transportation fuel demands while improving the energy efficiency of first generation biofuels.

Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity.

Switchgrass (Panicum virgatum L.) is a perennial C4 grass that is being developed as a bioenergy crop because it has high production yields and suitable agronomic traits. Five switchgrass biomass samples from upland and lowland switchgrass ecotypes harvested at different stages or maturity were used in this study. Switchgrass samples contained 317.0-385.0 g glucans/kg switchgrass dry basis (db) and 579.3-660.2 g total structural carbohydrates/kg switchgrass, db. Carbohydrate contents were greater for the upland ecotype versus lowland ecotype and increased with harvest maturity. Pretreatment of switchgrass with dilute ammonium hydroxide (8% w/w ammonium loading) at 170 degrees C for 20 min was determined to be effective for preparing switchgrass for enzymatic conversion to monosaccharides; glucose recoveries were 66.9-90.5% and xylose recoveries 60.1-84.2% of maximum and decreased with increased maturity at harvest. Subsequently, pretreated switchgrass samples were converted to ethanol by simultaneous saccharification and fermentation using engineered xylose-fermenting Saccharomyces cerevisiae strain YRH400. Ethanol yields were 176.2-202.01/Mg of switchgrass (db) and followed a similar trend as observed for enzymatic sugar yields.

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 dilute-acid 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.

Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification.

The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatment (dissolution and precipitation of cellulose by anti-solvent) switchgrass exhibited reduced cellulose crystallinity, increased surface area, and decreased lignin content compared to dilute acid pretreatment. Pretreated material was characterized by powder X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and chemistry methods. Ionic liquid pretreatment enabled a significant enhancement in the rate of enzyme hydrolysis of the cellulose component of switchgrass, with a rate increase of 16.7-fold, and a glucan yield of 96.0% obtained in 24h. These results indicate that ionic liquid pretreatment may offer unique advantages when compared to the dilute acid pretreatment process for switchgrass. However, the cost of the ionic liquid process must also be taken into consideration.

Plant species composition and biofuel yields of conservation grasslands.

Marginal croplands, such as those in the Conservation Reserve Program (CRP), have been suggested as a source of biomass for biofuel production. However, little is known about the composition of plant species on these conservation grasslands or their potential for ethanol production. Our objective was to assess the potential of CRP and other conservation grasslands for biofuel production, describing the relationships of plant species richness and tall native C4 prairie grass abundance with plant chemical composition and the resulting potential ethanol yield. We determined plant species composition and diversity at multiple scales with the modified Whittaker plot technique, aboveground biomass, plant chemical composition, and potential ethanol yield at 34 sites across the major ecological regions of the northeastern USA. Conservation grasslands with higher numbers of plant species had lower biomass yields and a lower ethanol yield per unit biomass compared with sites with fewer species. Thus, biofuel yield per unit land area decreased by 77% as plant species richness increased from 3 to 12.8 species per m2. We found that, as tall native C4 prairie grass abundance increased from 1.7% to 81.6%, the number of plant species decreased and aboveground biomass per unit land area and ethanol yield per unit biomass increased resulting in a 500% increased biofuel yield per unit land area. Plant species richness and composition are key determinants of biomass and ethanol yields from conservation grasslands and have implications for low-input high-diversity systems. Designing systems to include a large proportion of species with undesirable fermentation characteristics could reduce ethanol yields.

Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass.

Auto-fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1-n-ethyl-3-methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter- and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti-solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval.

Cell-wall composition and accessibility to hydrolytic enzymes is differentially altered in divergently bred switchgrass (Panicum virgatum L.) genotypes.

The aims of this study were to understand the genotypic variability in cell-wall composition and cell-wall accessibility to enzymes in select switchgrass plants obtained from two different populations derived from a base population of octaploid cultivars. Population C+3 was developed by three breeding generations for high digestibility and population C-1 developed by one generation of breeding for low digestibility. Above-ground biomass from 12 selected genotypes, three each with high or low digestibility within each population, was analyzed for their cell-wall aromatics and polysaccharides. The ratio of p-coumaric acid/ferulic acid was greater (P < or = 0.05) for the high-lignin C-1 population over the low-lignin C+3 population, although the amounts of these two phenolics did not differ between populations. Combined values of guaiacyl + syringyl-lignin were consistently higher in genotypes from the C-1 population as compared to the genotypes from the C+3 population. Overall, p-coumaric acid was released by enzymes in greater amounts than ferulic acid in all these genotypes. Genotypes in the C-1 population exhibited lower dry weight loss as compared to the genotypes in the C+3 population after enzymatic digestion, suggesting changes in cell-wall architecture. Overall, our data highlight the phenotypic plasticity coded by the switchgrass genome and suggest that combining dry matter digestibility with other more specific cell-wall traits could result in genotypes with greater utility as bioenergy feedstocks.

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels.

This review focuses on the potential advantages and disadvantages of forages such as switchgrass (Panicum virgatum), and two small grains: sorghum (Sorghum bicolor), and wheat (Triticum aesitvum), as feedstocks for biofuels. It highlights the synergy provided by applying what is known from forage digestibility and wheat and sorghum starch properties studies to the biofuels sector. Opportunities therefore, exist to improve biofuel qualities in these crops via genetics and agronomics. In contrast to cereal crops, switchgrass still retains tremendous exploitable genetic diversity, and can be specifically improved to fit a particular agronomic, management, and conversion platform. Combined with emerging studies on switchgrass genomics, conversion properties and management, the future for genetic modification of this species through conventional and molecular breeding strategies appear to be bright. The presence of brown-midrib mutations in sorghum that alter cell wall composition by reducing lignin and other attributes indicate that sorghum could serve as an important model species for C(4)-grasses. Utilization of the brown-midrib traits could lead to the development of forage and sweet sorghums as novel biomass crops. Additionally, wheat crop residue, and wheat and sorghum with improved starch content and composition represent alternate biofuel sources. However, the use of wheat starch as a biofuel is unlikely but its value as a model to study starch properties on biofuel yields holds significant promise.

Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum. L).

This work examined cell composition gradients in maturing tillers of switchgrass (Panicum virgatum L.) with the aim of developing baseline information on this important forage and biomass crop. Flowering tillers were collected from plants raised from seeds in a greenhouse and field, harvested at soil level and separated into internodes beginning with the node subtending the peduncle. Internodes were analyzed using microscopy, by fiber digestion, high-performance liquid chromatography and by gas chromatography-mass spectrometry to obtain anatomical and compositional data. Microscopy demonstrated the development and maturation of cortical fibers which eventually became confluent with the fiber sheath surrounding vascular bundles in the lower internodes. Detergent fiber analysis indicated increasing cellulose and lignin contents and decreases in cell solubles and hemicelluloses with increasing distance of the internodes from the top of the plant. Soluble phenolics were greatest in amounts and complexity in top internodes. The lower internodes contained greater levels of wall-bound phenolic acids, principally as 4-coumarate and ferulate.

Analysis of expressed sequence tags and the identification of associated short tandem repeats in switchgrass.

Switchgrass is a large, North American, perennial grass that is being evaluated as a potential energy crop. Expressed sequence tags (ESTs) were generated from four switchgrass cv. "Kanlow" cDNA libraries to create a gene inventory of 7,810 unique gene clusters from a total of 11,990 individual sequences. Blast similarity searches to SwissProt and GenBank non-redundant protein and nucleotide databases were performed and a total of 79% of these unique clusters were found to be similar to existing protein or nucleotide sequences. Tentative functional classification of 61% of the sequences was possible by association with appropriate gene ontology descriptors. Significant differential representation between genes in leaf, stem, crown, and callus libraries was observed for many highly expressed genes The unique gene clusters were screened for the presence of short tandem repeats for further development as microsatellite markers. A total of 334 gene clusters contained repeats representing 3.8% of the ESTs queried.