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At least 253 records · Page 14

Production of deuterated switchgrass by hydroponic cultivation

Deuterium enrichment of biological materials can potential enable expanded experimental use of small angle neutron scattering (SANS) to investigate molecular structural transitions of complex systems such as plant cell walls. Two key advances have been made that facilitate cultivation of switchgrass, an important forage and biofuel crop, for controlled isotopic enrichment: (1) perfusion system with individual chambers and (2) hydroponic growth from tiller cuttings. Plants were grown and maintained for several months with periodic harvest. Photosynthetic activity was monitored by measurement of CO 2 in outflow from the growth chambers. Plant morphology and composition appeared normal compared to matched controls grown with H 2 O. Using this improved method, gram quantities of switchgrass leaves and stems were produced by continuous hydroponic cultivation using growth medium consisting of basal mineral salts in 50% D 2 O. Deuterium incorporation was confirmed by detection of the O-D and C-D stretching peaks with FTIR and quantified by 1 H- and 2 H-NMR. Lastly, this capability to produce deuterated lignocellulosic biomass under controlled conditions will enhance investigation of cell wall structure and its deconstruction by neutron scattering and NMR techniques.

09 BIOMASS FUELS↗

Nitrogen Fertilizer, Arbuscular Mycorrhizal Fungi, and Soil Nematodes Affect Lignin Quality and Quantity in Switchgrass (Panicum virgatum L.)

Plant lignin content and composition, which limit cell wall digestibility and efficiency of cellulose conversion to bioethanol, can be influenced by belowground biotic and abiotic factors. Switchgrass (Panicum virgatum L.) is a leading lignocellulosic biofuel crop and forms strong belowground associations with arbuscular mycorrhizal fungi (AMF), is susceptible to belowground plant-parasitic nematodes (PPN), and when grown in monoculture generally requires nitrogen (N) fertilization. The main objectives of the study were to investigate the effects of N fertilizer and belowground organisms on lignin content and composition of switchgrass. Here, leaf, stem, and root tissues were evaluated separately to test whether these factors had varying belowground (local) or aboveground (systemic) effects on plants. These factors were manipulated in a field study in 2017 using biocide applications to reduce soil fungi and nematodes. Combined biocide application reduced p-hydroxyphenyl (H) unit abundance in the leaves by 14% and increased the syringyl:guaiacyl (S:G) ratio in stems by 2%. Application of fungicide alone increased stem syringyl (S) unit by 12.4% as compared with control plots, and 11.1% as compared with nematicide plots. Overall, fertilizer increased total stem lignin by 3%, stem S unit by 6.7%, and stem S:G ratio by 10%, whereas it reduced the amount of H-unit in the roots by 11%. While the effects of N fertilizer were more pronounced in this study, changes to soil organisms had similar magnitudes of effect for some measures of lignin, indicating that these belowground interactions may be important for growers to consider in the future.

59 BASIC BIOLOGICAL SCIENCES↗

Nth-plant scenario for blended pellets of Miscanthus, Switchgrass, and Corn Stover using multi-modal transportation: Biorefineries and depots in the contiguous U.S.

The sustainability of the biofuel industry depends on the development of a mature conversion technology on a national level that can take advantage of the economies of scale: the nth-plant. Here, this study addresses the logistic challenge of mobilizing national cellulosic feedstock supplies for a sustainable bioenergy industry. A Mixed Integer Linear Programming (MILP) model was developed and updated to deliver on-spec biomass that considers both a desired quantity and quality at the biorefinery. Our supply chain analysis includes multi-modal transport (truck and rail), varying depot and biorefinery sizes, and feedstock blends of corn stover (harvested by either a two- or three-pass method), switchgrass, and miscanthus. The following US states: Illinois, Kansas, Missouri, North Carolina, Oklahoma, Georgia, and Texas were identified as key locations for producing accessible miscanthus. Based on our most optimistic scenario, using trucks as the only transportation mode in 2040 with a cost target of $\$$79/dt, corn stover, switchgrass, and miscanthus could help meet 48% of the EPA target, 173 million dry tons that translate into 7.8 billion GGE. The addition of rail transportation for biomass delivery to biorefineries could help meet 79% of the EPA target, 283 million dry tons that translate into 12.7 billion GGE.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

Genetic determinants of switchgrass-root-associated microbiota in field sites spanning its natural range

A fundamental goal in plant microbiome research is to determine the relative impacts of host and environmental effects on root microbiota composition, particularly how host genotype impacts bacterial community composition. Most studies characterizing the effect of plant genotype on root microbiota undersample host genetic diversity and grow plants outside of their native ranges, making the associations between host and microbes difficult to interpret. Here, we characterized the root microbiota of a large diversity panel of switchgrass, a North American native C4 bioenergy crop, in three field locations spanning its native range. Our data, composed of 1,961 samples, suggest that field location is the primary determinant of microbiome composition; however, substantial heritable variation is widespread across bacterial taxa, especially those in the Sphingomonadaceae family. Despite diverse compositions, relatively few highly prevalent taxa make up the majority of the switchgrass root microbiota, a large fraction of which is shared across sites. Local genotypes preferentially recruit/filter for local microbes, supporting the idea of affinity between local plants and their microbiota. Using genome-wide association, we identified loci impacting the abundance of >400 microbial strains and found an enrichment of genes involved in immune responses, signaling pathways, and secondary metabolism. We found loci associated with over half of the core microbiota (i.e., microbes in >80% of samples), regardless of field location. Finally, we show a genetic relationship between a basal plant immunity pathway and relative abundances of root microbiota. Furthermore, this study brings us closer to harnessing and manipulating beneficial microbial associations via host genetics.

54 ENVIRONMENTAL SCIENCES↗

Effect of in Vivo Deuteration on Structure of Switchgrass Lignin

Biomass deuteration is an effective engineering method that can be used to provide key insights into understanding of biomass recalcitrance and the complex biomass conversion process. In this study, production of deuterated switchgrass was accomplished by growing the plants in 50% D 2 O under hydroponic conditions in a perfusion chamber. Cellulolytic enzyme lignin was isolated from deuterated switchgrass, characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) and compared with its protiated control sample to determine the effect of in vivo deuteration on the chemical structure of lignin. FTIR results showed that D 2 O can be taken up by the roots and transported to the leaves, and deuterium was subsequently incorporated into hydroxyl and alkyl groups in the plant and its lignin through photosynthesis. According to GPC results, deuterated lignin had slightly higher molecular weight, presumably due to isotope effects. 31 P and heteronuclear single quantum coherence (HSQC) NMR results revealed that lignin in the deuterated biomass preserved its native physicochemical characteristics. Finally, the conserved characteristics of the deuterated lignin show its great potential applications for structural and dynamic studies of lignocellulose by techniques such as neutron scattering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Silicon cantilever functionalization for cellulose-specific chemical force imaging of switchgrass

A method for direct functionalization of silicon and silicon nitride cantilevers with bifunctional silanes was tested with model surfaces to determine adhesive forces for different hydrogen-bonding chemistries. Application for biomass surface characterization was tested by mapping switchgrass and isolated switchgrass cellulose in topographic and force-volume mode using a cellulose-specific cantilever.

60 APPLIED LIFE SCIENCES↗

Genomic prediction of switchgrass winter survivorship across diverse lowland populations

Abstract In the North-Central United States, lowland ecotype switchgrass can increase yield by up to 50% compared with locally adapted but early flowering cultivars. However, lowland ecotypes are not winter tolerant. The mechanism for winter damage is unknown but previously has been associated with late flowering time. This study investigated heading date (measured for two years) and winter survivorship (measured for three years) in a multi-generation population generated from two winter-hardy lowland individuals and diverse southern lowland populations. Sequencing data (311,776 markers) from 1,306 individuals were used to evaluate genome-wide trait prediction through cross-validation and progeny prediction (n = 52). Genetic variance for heading date and winter survivorship was additive with high narrow-sense heritability (0.64 and 0.71, respectively) and reliability (0.68 and 0.76, respectively). The initial negative correlation between winter survivorship and heading date degraded across generations (F1 r = −0.43, pseudo-F2 r = −0.28, pseudo-F2 progeny r = −0.15). Within-family predictive ability was moderately high for heading date and winter survivorship (0.53 and 0.52, respectively). A multi-trait model did not improve predictive ability for either trait. Progeny predictive ability was 0.71 for winter survivorship and 0.53 for heading date. These results suggest that lowland ecotype populations can obtain sufficient survival rates in the northern United States with two or three cycles of effective selection. Despite accurate genomic prediction, naturally occurring winter mortality successfully isolated winter tolerant genotypes and appears to be an efficient method to develop high-yielding, cold-tolerant switchgrass cultivars.

60 APPLIED LIFE SCIENCES↗

Optimizing genomic prediction for complex traits via investigating multiple factors in switchgrass

Genomic prediction has accelerated breeding processes and provided mechanistic insights into the genetic bases of complex traits. To further optimize genomic prediction, we assess the impact of genome assemblies, genotyping approaches, variant types, allelic complexities, polyploidy levels, and population structures on the prediction of 20 complex traits in switchgrass (Panicum virgatum L.), a perennial biofuel feedstock. Surprisingly, short read-based genome assembly performs comparably to or even better than long read-based assembly. Due to higher gene coverage, exome capture and multi-allelic variants outperform genotyping-by-sequencing and bi-allelic variants, respectively. Tetraploid models show higher prediction accuracy than octoploid models for most traits, likely due to the greater genetic distances among tetraploids. Depending on the trait in question, different types of variants need to be integrated for optimal predictions. Furthermore, our study provides insights into the factors influencing genomic prediction outcomes, guiding best practices for future studies and for improving agronomic traits in switchgrass and other species through selective breeding.

60 APPLIED LIFE SCIENCES↗

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation

Abstract Knowing how switchgrass ( Panicum virgatum L . ) responds and adapts to phosphorus (P)‐limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P‐stress on genome transcription and whole‐plant metabolism, physiology and development in switchgrass. P‐limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P‐stress, and decreased root diameter with increased density and length of root hairs at severe P‐stress. RNA‐seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P‐stress in roots and/or shoots compared to P‐replete plants, with many stress‐induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P‐acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P‐stress in shoots and roots, although this trend reversed under severe P‐stress, especially in shoots.

59 BASIC BIOLOGICAL SCIENCES↗

Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.

09 BIOMASS FUELS↗

Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii

ABSTRACT Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(–) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.

60 APPLIED LIFE SCIENCES↗

Transcriptome analysis in switchgrass discloses ecotype difference in photosynthetic efficiency

Background: Switchgrass, a warm-season perennial grass studied as a potential dedicated biofuel feedstock, is classified into two main taxa – lowland and upland ecotypes – that differ in morphology and habitat of adaptation. But there is limited information on their inherent molecular variations. Results: Transcriptome analysis by RNA-sequencing (RNA-Seq) was conducted for lowland and upland ecotypes to document their gene expression variations. Mapping of transcriptome to the reference genome (Panicum virgatum v1. 1) revealed that the lowland and upland ecotypes differ substantially in sets of genes transcribed as well as levels of expression. Differential gene expression analysis exhibited that transcripts related to photosynthesis efficiency and development and photosystem reaction center subunits were upregulated in lowlands compared to upland genotype. On the other hand, catalase isozymes, helix-loop-helix, late embryogenesis abundant group I, photosulfokinases, and S-adenosyl methionine synthase gene transcripts were upregulated in the upland compared to the lowlands. At ≥100x coverage and ≥5% minor allele frequency, a total of 25,894 and 16,979 single nucleotide polymorphism (SNP) markers were discovered for VS16 (upland ecotype) and K5 (lowland ecotype) against the reference genome. The allele combination of the SNPs revealed that the transition mutations are more prevalent than the transversion mutations. Conclusions: The gene ontology (GO) analysis of the transcriptome indicated lowland ecotype had significantly higher representation for cellular components associated with photosynthesis machinery controlling carbon fixation. In addition, using the transcriptome data, SNP markers were detected, which were distributed throughout the genome. The differentially expressed genes and SNP markers detected in this study would be useful resources for traits mapping and gene transfer across ecotypes in switchgrass breeding for increased biomass yield for biofuel conversion.

09 BIOMASS FUELS↗

Data for Responsiveness of Miscanthus and Switchgrass Yields to Stand Age and Nitrogen Fertilization: A Meta-regression Analysis

The compiled datasets include plot level observations of energy crops (miscanthus and switchgrass) from recent experimental field trials in the US including dry biomass yield, location, state, region, harvest year, growing season degree days (GDD), winter season heating degree days (HDD), growing season cumulative precipitation, annual nitrogen application rate, age of the pant when harvested, National Commodity Crop Productivity Index (NCCPI) values, and cultivar type (switchgrass) from various published and unpublished sources. The stata codes include estimation procedures for four different specifications, i.e., Model A includes deterministic effect without interaction terms; Model B includes deterministic effect with interaction terms (N2, age2, N × age, GDD2, precip2, N × NCCPI); Model C includes deterministic effect with interaction terms, study, and location random effect; Model D includes deterministic effect with interaction terms, harvest year augmented study, and location random effect.

Age↗

Influence of Weather on the Predicted Moisture Content of Field Chopped EnergySorghum and Switchgrass

To determine the effects of weather on harvested moisture content (MC) of switchgrass (Panicum virgatum) and energy sorghum (Sorghum bicolor), tracking of harvest progress on individual fields in the Integrated Biomass Supply and Logistics (IBSAL) model was modified to allow: i) rewetting of swathed material in the drying formulae; and ii) field queuing rules based on equipment availability and weather. Estimated crop yield and initial MC by harvest date, as observed in field trials, along with the modeling of different delays between mowing and harvest allowed estimation of harvested MC, annual tonnage processed and associated processing cost differences by crop and location over 10 years. Extending the hours of annual equipment use had minor implications on cost of production. Energy sorghum proved difficult to dry in the field. Its higher yield, leading to shorter supply distance to the plant, may justify harvesting of energy sorghum early in the season with drier weather. Lastly, later harvest for lower-yielding switchgrass offers MC advantages.

09 BIOMASS FUELS↗

Development of Kinetics and Mathematical Models for High-Pressure Gasification of Lignite-Switchgrass Blends: Cooperative Research and Development Final Report, CRADA Number CRD-11-447

NREL will work with Participant as a subtier partner under DE-FOA-0000240 titled "Co-Production of Power, Fuels, and Chemicals via Coal/Biomass Mixtures." The goal of the project is to determine the gasification characteristics of switchgrass and lignite mixtures and develop kinetic models. NREL will utilize a pressurized thermogravimetric analyzer to measure the reactivity of chars generated in a pressurized entrained-flow reactor at Participant's facilities and to determine the evolution of gaseous species during pyrolysis of switchgrass-lignite mixtures. Mass spectrometry and Fourier-transform infrared analysis will be used to identify and quantify the gaseous species. The results of the project will aid in defining key reactive properties of mixed coal biomass fuels.

09 BIOMASS FUELS↗

Unraveling the Genetics of Two Key Biomass Traits that Differentiate Upland and Lowland Tetraploid Switchgrass Ecotypes, Colonization by Mycorrhizal Fungi and Frost Tolerance

The overall objectives of the proposal were to dissect the genetics of the ability of switchgrass to interact with arbuscular mycorrhizal fungi (AMF) and to tolerate subzero temperatures, and to investigate the effect of AMF colonization on freezing tolerance and biomass production. In order to achieve this, we generated genetic maps in an F2 mapping population derived from a cross between the lowland genotype AP13 and the upland genotype VS16. In addition to providing a framework for identification of quantitative trait loci (QTL), the genetic maps were used to validate and improve the switchgrass genome assembly. We mapped phenotypic quantitative trait loci (QTL) for AMF colonization and cold tolerance in the F 2 population. We demonstrated that identification of QTL for AMF colonization was achieved only if AMF genus-specific colonization levels were used as phenotypes. No QTL were obtained when the overall level of AMF colonization was used as a trait. We conducted RNASeq on a subset of the F2 progeny following cold acclimation, and developed statistical methods to conduct expression QTL (eQTL) analyses in a polyploid organism. A preliminary analysis of the results identified eQTL for a range of genes that were differentially regulated in the lowland AP13 compared to the upland VS16 under cold acclimation. Once sequencing of the transcriptomes of a temporal replicate of the mapping population that was cold-acclimated using the same conditions has been completed, a final eQTL analysis will be conducted.

09 BIOMASS FUELS↗

Drought adaptation index (DAI) based on BLUP as a selection approach for drought-resilient switchgrass germplasm

This study introduces a Drought Adaptation Index (DAI), derived from Best Linear Unbiased Prediction (BLUP), as a method to assess drought resilience in switchgrass (Panicum virgatum L.). A panel of 404 genotypes was evaluated under drought-stressed (CV) and well-watered (UC) conditions over four consecutive years (2019–2022). BLUP-estimated biomass yields were used to calculate the DAI, which enabled classification of genotypes into four adaptation groups: very well-adapted, well-adapted, adapted, and unadapted. The DAI was compared with conventional drought tolerance indices, including the Stress Susceptibility Index (SSI), Stress Tolerance Index (STI), Geometric Mean Productivity (GMP), and Yield Stability Index (YSI). Correlation analyses demonstrated strong agreement between DAI and these indices, supporting its validity and consistency. Biplot analyses using the Genotype plus Genotype-by-Environment Interaction (GGE) and Additive Main Effects and Multiplicative Interaction (AMMI) models revealed significant genotype-by-environment interactions (GEI) and identified J222.A, J463.A, and J295.A. A as high-performing genotypes, with J222.A exhibiting greater yield stability across treatments and years. Additionally, DAI isoline curves provided a graphical representation of differential genotype performance under drought and control conditions. These visualizations aided in distinguishing genotypes with stable and superior biomass yield across contrasting environments. Overall, the BLUP-based DAI is a robust and practical selection tool that improves the accuracy of identifying drought-resilient, high-yielding switchgrass genotypes. Its integration into breeding programs offers a comprehensive framework for improving biomass productivity and stress adaptation under variable climatic conditions. The application of DAI supports the development of climate-resilient cultivars and contributes to sustainable bioenergy and forage production systems.

BLUP↗

Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-Mimic Cell Death

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, has been shown to alter lignification and increase biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediates C1 metabolism and provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would impact lignification even more than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding was that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes related to oxidative and defense responses. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergistic effects on agronomic performance.

methylenetetrahydrofolate reductase (MTHFR)↗