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Reductions in either lignin concentration or ferulate cross-linking have been shown to be independent mechanisms for increasing herbage digestibility by increasing the enzymatic degradability of cell-wall polysaccharides. However, both of these traits are frequently associated with reductions in agronomic fitness, including dwarfing, developmental abnormalities, and loss of persistence. The objective of this study was to evaluate the impacts of selection for these two traits on agronomic fitness of three temperate perennial grasses: orchardgrass (Dactylis glomerate L.), reed canarygrass (Phalaris arundinacea L.), and smooth bromegrass (Bromus inermis Leyss.). All possible crosses were made within three groups of eight parents, with parents selected to represent four divergent phenotypes: high or low etherified ferulates and high or low Klason lignin. Progeny were evaluated for forage yield under a three-cut management and for tiller density and ground cover under a frequent-harvest management. Selection for reduced lignin concentration had highly variable and inconsistent impacts on agronomic fitness, varying widely among species. Conversely, selection for reduced ferulate cross-linking resulted in a general decline for all measures of agronomic fitness within each species. The results support the hypothesis put forward from an earlier study and from the literature that reduced ferulate cross-linking results in significant impairments to stem development of these temperate grasses.

Additional file of Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

Additional file 26:Optimization of de novo assembly of Illumina GA-II EST reads with a series of k -mers using the Velvet program[32]. A figure showing the median sequence length of the contigs (y-axis) for a series of k-mers (31, 37, 41, 47, 51, 57, 61, 63, 65) tested using the Velvet program. k-mer 61 produced the longest median sequence length. (PDF 64 KB)

Authors’ original file for figure 6

Additional file 7:Functional classes over- or under-represented among SNP-harboring genes. A figure showing the functional class over-representation analysis conducted for SNP-harboring genes. Functional classes that are over- or under-represented among SNP-harboring genes were identified using the PageMan over-representation analysis module. The z-vlaues for significant classes identified after Fisher's exact test with Bonferroni correction (z-value cutoff of 1) were false color coded using a scale of -5 to +5. The intensity of blue and red indicate the degree of over- and under-representation of the corresponding class, respectively. (PDF 127 KB)

Additional file 13:Functional classes over- or under-represented among the top 500 most abundant transcripts in each library. A figure showing the results from functional class over-representation analysis for the top 500 most abundant transcripts in ES and PES internodes of alfalfa genotypes 708 and 773. For details, see the description for additional file 7. (PDF 404 KB)

Maize (Zea mays L.) stover and cobs are potential feedstock sources for cellulosic ethanol production. Nitrogen (N) fertilization is an important management decision that influences cellulosic biomass and grain production, but its effect on cell wall composition and subsequent cellulosic ethanol production is not known. The objectives of this study were to quantify the responses of maize stover (leaves, stalks, husks, and tassel) and cob cell wall composition and theoretical ethanol yield potential to N fertilization across a range of sites. Field experiments were conducted at rainfed and irrigated sites in Minnesota, USA, over a 2-year period. Stover cell wall polysaccharides, pentose sugar concentration, and theoretical ethanol yield decreased as N fertilization increased. Stover Klason lignin increased with N fertilization at all sites. Cob cell wall composition was less sensitive to N fertilization, as only pentose and Klason lignin decreased with N fertilization at two and one site(s), respectively, and hexose increased with N fertilization at one of eight sites. Cob theoretical ethanol yield was not affected by N fertilization at any site. These results indicate variation in stover cellulosic ethanol production is possible as a result of N management. This study also demonstrated that cell wall composition and subsequent theoretical ethanol yield of maize cobs are generally more stable than those with stover because of overall less sensitivity to N management.

Dietary fiber supplements are used to manage fecal incontinence (FI), but little is known about the fiber type to recommend or the level of effectiveness of such supplements, which appears related to the fermentability of the fiber. The aim of this single-blind, randomized controlled trial was to compare the effects of three dietary fiber supplements (carboxymethylcellulose [CMC], gum arabic [GA], or psyllium) with differing levels of fermentability to a placebo in community-living individuals incontinent of loose/liquid feces. The primary outcome was FI frequency; secondary outcomes included FI amount and consistency, supplement intolerance, and quality of life (QoL). Possible mechanisms underlying supplement effects were also examined. After a 14-day baseline, 189 subjects consumed a placebo or 16 g total fiber/day of one of the fiber supplements for 32 days. FI frequency significantly decreased after psyllium supplementation versus placebo, in both intent-to-treat and per-protocol mixed model analyses. CMC increased FI frequency. In intent-to-treat analysis, the number of FI episodes/week after supplementation was estimated to be 5.5 for Placebo, 2.5 for Psyllium, 4.3 for GA, and 6.2 for CMC. Only psyllium consumption resulted in a gel in feces. Supplement intolerance was low. QoL scores did not differ among groups. Patients with FI may experience a reduction in FI frequency after psyllium supplementation, and decreased FI frequency has been shown to be an important personal goal of treatment for patients with FI. Formation of a gel in feces appears to be a mechanism by which residual psyllium improved FI.

An alfalfa ( Medicago sativa L) biomass energy production system would produce two products. Leaves would be separated from stems to produce a protein feed for livestock while stems would be processed to produce ethanol. Therefore, maximum yields of both leaves and stems are essential for profitability of this biomass production system. Our objective was to evaluate the impact of growth environment (locations, years and plant density) and harvest maturity stage (early bud (4 annual cuts) and late flower (3 annual cuts)) on leaf crude protein and potential ethanol yields for four alfalfa germplasms, two with high forage quality, and two non-lodging biomass types. Potential ethanol yield was greater at late flower compared to early bud, while leaf crude protein concentration was similar at the two harvest maturity stages at both locations. Leaf crude protein yield was greater at the Minnesota (MN) site compared to Wisconsin (WI) site. The two non-lodging biomass germplasms had greater potential ethanol yield compared to the high forage quality cultivars in WI, but no differences among the alfalfa germplasms were found for ethanol yield at MN. In WI, no differences were found among the germplasms for leaf crude protein yield, but the high quality cultivars had greater leaf crude protein yield than the non-lodging germplasms in MN. While germplasm differences were found for leaf crude protein and potential ethanol yields, the environmental influences of harvest date and locations had the greatest impact on these two alfalfa biomass energy products.

Although clinical benefits of dietary fiber supplementation seem to depend partially on the extent of fiber degradation and fermentation by colonic bacteria, little is known about the effect of supplemental fiber type on bacterial metabolism. In an experiment using a nonadapted human bacterial population from three normal subjects, the extent of in vitro fermentation was greater for gum arabic (GA) than for psyllium (PSY), which was greater than that for carboxymethylcellulose (CMC). In a separate experiment, in vitro incubation with feces from 52 subjects with fecal incontinence, before and after random assignment to and consumption of one of three fiber (GA, PSY, or CMC) supplements or a placebo for 20-21 days, indicated that prior consumption of a specific fiber source did not increase its degradation by fecal bacteria. Results suggest that the colonic microbial community enriched on a particular fiber substrate can rapidly adapt to the presentation of a new fiber substrate. Clinical implications of the findings are that intake of a fiber source by humans is not expected to result in bacterial adaptation that would require continually larger and eventually intolerable amounts of fiber to achieve therapeutic benefits.

Biomass composition of switchgrass (Panicum virgatum L.) can affect its utilization by ruminants and its conversion to liquid fuels in a biorefinery. The objective of this study was to evaluate the effects of six generations of divergent breeding for forage in vitro dry matter digestibility (IVDMD) on switchgrass biomass composition, forage quality traits, and ethanol yield. Initially there was one cycle of selection for both low (C-1) and high IVDMD (C1 = cv. Trailblazer), followed by four additional breeding cycles for high IVDMD. In cycles 4 and 5, winter survival was included as a selection criterion because of decreased winter survival of the C3 population. The experimental populations that were produced by these breeding generations and nine half-sib families from cycle 5 were evaluated for two post-establishment years at the research station in eastern Nebraska, where all the breeding work was conducted. The six breeding generations resulted in significant differences among the populations for all the 28 cell wall and non-cell-wall composition variables measured, forage quality, and ethanol yield traits measured except for total biomass C, cell wall concentration, soluble glucose, and etherified ferulates. These traits included all cell wall and nonstructural carbohydrates. Breeding for the heritable complex trait IVDMD affected a large number of plant biomass characteristics and also adversely affected plant biomass yield and winter survival. K.P. Vogel, R.B. Mitchell, and G. Sarath, USDA-ARS, Grain, Forage, and Bioenergy Res. Unit, 137 Keim Hall, Dep. of Agronomy and Horticulture, Univ. of Nebraska-Lincoln, Lincoln, NE 68583-0937; H.G. Jung, USDA-ARS (retired), Plant Science Research Unit and Dep. Agronomy and Plant Genetics, 411 Borlaug Hall, 1991, Upper Buford Circle, St. Paul, MN 55108-6026; B.S. Dien, USDA-ARS Fermentation Biotechnology Res. Unit, National Center for Agricultural Utilization Res., 1815 N. University Street, Peoria, IL 61604; M.D. Casler, USDA-ARS, U.S. Dairy Forage Res. Center, 1925 Linden Drive West, Madison, WI 53706-1108. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendations or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity employer. Received 18 Sept. 2012. *Corresponding author (Ken.Vogel@ars.usda.gov). Abbreviations: ADL, reduced acid detergent lignin; BW IVDMD, in vitro dry matter digestibility; MSFS, multi-step family selection; NIRS, near infrared reflectance spectrometry; RRPS, restricted recurrent phenotypic selection; SSF, simultaneous saccharification and fermentation; see Table 2 for an extensive listing of abbreviations for biomass composition or conversion traits. Published in Crop Sci. 53:853–862 (2013). doi: 10.2135/cropsci2012.09.0542 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

The harvest of corn (Zea mays L.) stover and cobs for cellulosic ethanol production will likely accelerate the depletion of soil N, P, K, and S, thus increasing nutrient replacement costs. Fertilizer N rate is a management variable that may influence N, P, K, and S removed by corn cellulosic materials. The effects of fertilizer N rate on grain and cellulosic biomass yields have been reported for the Upper Midwest, but no information exists regarding its effects on stover and cob nutrient removal across a range of rates in diverse environments. Experiments were conducted in eight environments in Minnesota to determine the effect of fertilizer N rate on N, P, K, and S removal by corn stover (leaves, stalks, husks, shanks, and tassels) and cobs. Removal of N, K, and S by stover and cobs generally increased as fertilizer N rate increased, although the response often differed among environments. Removal of P by stover and cobs decreased or did not change as fertilizer N rate increased. Harvesting 7.5 Mg ha⁻¹ of corn stover removed on average 46, 3.5, 76, and 3.7 kg ha⁻¹ of N, P, K, and S, respectively, while harvesting 1.5 Mg ha⁻¹ of cobs removed 6.1, 0.37, 9.5, and 0.38 kg ha⁻¹ of N, P, K, and S, respectively. The harvest of solely corn cobs would remove 9 to 12% less N, P, K, and S, and thus deplete soil nutrient pools at slower rates compared with the harvest of both stover and cobs.

Genomewide selection (GWS) is markerassisted selection without identifying markers with signifi cant effects. Our previous work with the intermated B73 × Mo17 maize (Zea mays L.) population revealed signifi cant variation for grain yield and stover-quality traits important for cellulosic ethanol production. Our objectives were to determine (i) if realized gains from selection are larger with GWS than with markerassisted recurrent selection (MARS), which involves selection for markers with signifi cant effects; and (ii) how multiple traits respond to multiple cycles of GWS and MARS. In 2007, testcrosses of 223 recombinant inbreds developed from B73 × Mo17 (Cycle 0) were evaluated at four Minnesota locations and genotyped with 287 single nucleotide polymorphism markers. Individuals with the best performance for a Stover Index and a Yield + Stover Index were intermated to form Cycle 1. Both GWS and MARS were then conducted until Cycle 3. Multilocation trials in 2010 indicated that gains for the Stover Index and Yield + Stover Index were 14 to 50% larger (signifi cant at P = 0.05) with GWS than with MARS. Gains in individual traits were mostly nonsignifi cant. Inbreeding coeffi cients ranged from 0.28 to 0.38 by Cycle 3 of GWS and MARS. For stover-quality traits, correlations between wet chemistry and near-infrared refl ectance spectroscopy predictions decreased after selection. We believe this is the fi rst published report of a GWS experiment in crops, and our results indicate that using all available markers for predicting genotypic value leads to greater gain than using a subset of markers with signifi cant effects. J.M. Massman and R. Bernardo, Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Cir., Saint Paul, MN 55108; H.-J.G. Jung, USDA-ARS Plant Science Research Unit, 411 Borlaug Hall, 1991 Upper Buford Cir., Saint Paul, MN 55108. Received 17 Feb. 2012. *Corresponding author (bernardo@umn.edu). Abbreviations: GWS, genomewide selection; MARS, marker-assisted recurrent selection; NIRS, near-infrared refl ectance spectroscopy; QTL, quantitative trait loci; RR-BLUP, ridge regression–best linear unbiased prediction; SNP, single nucleotide polymorphism. Published in Crop Sci. 53:1–9 (2013). doi: 10.2135/cropsci2012.02.0112 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Alfalfa has considerable potential as a feedstock for production of fuels, feed, and industrial materials. Alfalfa breeders have utilized the extensive germplasm resources of alfalfa to introduce disease and insect resistance, expand environmental adaptation, and improve forage quality. Most improvements in forage quality of alfalfa have occurred through changes in harvest management and production practices. The yield of potentially fermentable sugars from the conversion process is the critical response variable in assessing the value of alfalfa as an ethanol production feedstock. In addition to production of proteins, the use of transgenic alfalfa to produce other industrial feed stocks has been explored. The production costs of alfalfa are low and cultivation of the crop has numerous environmental benefits. A biomass-type of alfalfa is being developed that is more upright in growth habit and performs well in a reduced frequency harvest management system, maximizing the yield of both leaf and stem fractions while lowering production costs.

Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (CLs). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8–8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged., This research was supported by the National Plan for Research and Development of Spain (Projects Cod. AGL2006-13140, AGL 2009-09611). J. Barros-Rios acknowledges a grant from the Ministry of Science and Innovation (Spain). R. Santiago acknowledges a postdoctoral contract “Isidro Parga Pondal” financed by the Autonomous Government of Galicia (Spain) and the European Social Fund., MICINN, Xunta de Galicia, UE

Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter Published by Elsevier Ireland Ltd.

This study was undertaken to identify tissue-specific biochemical traits that may be targeted in breeding programs for improving forage digestibility. We compared cell wall chemical composition and 24- and 96-h in vitro degradabilities in separated pith and rind tissues of lower stem internodes from six maize (Zea mays L.) inbred lines. Across genotypes, rind tissues had higher total cell wall and ether-linked ferulate concentrations (69%, and 53% higher, respectively), were less degradable, and the increased degradation between incubation times was larger than in pith tissues. Genotypes exhibited genetic variation for glucose, xylose and hydroxinnamate ester concentrations and for most measures of cell wall degradability. However, none of genotypes had extreme values for these cell wall traits in both tissues, indicating that regulation of cell wall characteristics may be tissue-independent. Cross-linking of lignin to arabinoxylan through ferulate ethers explained a portion of cell wall degradability variation in both tissues (R 2=0.55 and 0.24 of pith and rind cell wall 24-h degradability, respectively), and in conjunction with Klason lignin concentration appeared to be one of the major determinants of cell wall degradability. p-Coumarate ester and glucose concentrations were the overall best predictors of pith cell wall degradability, and arabinose concentration was a good indicator of rapid rind cell wall degradability, suggesting the potential usefulness of these cell wall traits in breeding programs to improve cell wall digestibility. However, the majority of rind cell wall degradability variation (R 2=0.65 and 0.73 of 24- and 96-h degradabilities, respectively) was not explained by the cell wall traits evaluated. Additional biochemical traits and anatomical factors influence cell wall degradation in isolated tissues. © 2012 Elsevier B.V., MICINN-National Plan for Research and Development of Spain (Projects Cod. AGL2006-13140, AGL 2009-09611); Xunta de Galicia-postdoctoral contract “Isidro Parga Pondal”

Published in Agron. J. 104:363–370 (2012) Posted online 11 Jan 2012 doi:10.2134/agronj2011.0279 Copyright © 2012 by the American Society of Agronomy, 5585 Guilford Road, Madison, WI 53711. All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. T Energy Independence and Security Act of 2007 mandates that the United States produce 136 billion liters per year of renewable transportation fuel from both starch and cellulosic sources by 2022 (USDA, 2010). Corn grain will likely continue to be the primary feedstock for starch-based ethanol production, while the emerging cellulosic ethanol-based industry will rely on materials including, but not limited to, corn stover (the material remaining after grain harvest), switchgrass (Panicum virgatum L.), biomass (sweet) sorghum [(Sorghum bicolor (L.) Moench], sugarcane (Saccharum officinarum L.), and woody materials to meet future demands (Perlack et al., 2005; Sissine, 2007). Of these cellulosic sources, corn stover is projected to play an important role due to its high availability and production in close proximity to existing ethanol production facilities in the Midwest. Corn offers distinct benefits compared to other biofuel crops: production of both starch and cellulosic feedstocks and its total biomass is often greater than most other biofuel crop species. Work by Propheter et al. (2010) in Kansas showed that corn produced greater total biomass yields (starch and cellulosic combined) than switchgrass, Miscanthus (Miscanthus × giganteus), and big bluestem (Andropogon gerardii Vitman), and similar yields as several sorghum species. When only stover yields were compared, corn stover yield was similar to or greater than yields of nearly all perennial grass species. Based on a harvest index (the percentage of grain dry matter relative to total aboveground plant dry matter) of 0.45 suggested by Nielsen (1995), a grain yield of 7.0 Mg ha–1 would conservatively yield approximately 8.5 Mg ha–1 of cellulosic material. Using a harvest index of 0.50, Wilhelm et al. (2004) estimated that 137 Tg of cellulosic material from corn stover may be available in Illinois, Iowa, Minnesota, and Nebraska, which represented 54% of the total corn stover produced in the United States. James et al. (2010) concluded that for projected cellulosic material market values, corn biofuel systems are more profitable than systems including native grasses and perennial woody species. In addition to the greater biomass production and greater profitability, cropping systems containing corn are well established, while land devoted for the growth of other cellulosic sources, like switchgrass and Miscanthus, is currently limited. Nitrogen fertilization is a key component in corn grain production and economically optimum rates have been established in the U.S. Corn Belt, particularly Minnesota (Kaiser et al., 2011; Sawyer et al., 2006; Lamb and Nicolai, 2007). Biofuelbased agronomic research of corn has historically focused on grain (Coulter and Nafziger, 2008; Reicks et al., 2009; Hao et al., 2010; Wortmann et al., 2010) or on the effects of removing the stover on soil quality (Doran et al., 1984; Linden et al., 2000; Wilhelm et al., 2007; Blanco-Canqui, 2010), but generally not on stover and cob biomass production or ethanol yield. Varvel et al. (2008) reported the effects of N fertilization on stover production, showing that increasing N fertilization from 0 to 60 kg N ha–1 increased stover production. However, the study did not partition cobs from the stalk/leaf fraction, so it is not possible to quantify the effects of N fertilization on each individual fraction. Propheter et al. (2010) reported the total ethanol production ABSTRACT Corn (Zea mays L.) stover will likely play an integral role in near-term attempts to produce renewable cellulosic transportation fuels. However, little is known regarding the effect of N fertilization on biomass and ethanol yields of stover and cobs. The objectives were to evaluate the effect of N fertilization on stover and cob biomass and ethanol yields across a range of environments, and to determine if these biomass and ethanol yields can be maximized within N fertilization rates for grain yield optimization. Field experiments were conducted over eight diverse environments across Minnesota. Overall, stover and cob biomass and ethanol yields increased with increasing N fertilization, and agronomically optimum nitrogen rates (AONR) were identified in nearly all environments that were responsive to N fertilization. Ethanol yields for stover ranged from 2414 to 3842 L ha–1, whereas ethanol yields for cobs ranged from 513 to 906 L ha–1. When AONRs for stover and cob ethanol yields were compared to the respective AONR for grain yield, stover ethanol yield was maximized at N fertilization rates below the AONR for grain yield in four of the seven responsive environments, while cob ethanol yield was maximized at N fertilization rates below the AONR for grain yield in five of the six responsive environments. These results suggest that stover and cob ethanol yields will often be maximized when grain yield optimization is the primary goal.

Published in Agron. J. 104:54–64 (2012) Posted online 23 Nov 2011 doi:10.2134/agronj2011.0195 Copyright © 2012 by the American Society of Agronomy, 5585 Guilford Road, Madison, WI 53711. All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. C refineries will require substantial amounts of biomass on a year-round basis and are expected to have higher capital costs than similar sized grain ethanol plants based on first-generation biomass refining technology (Wright and Brown, 2007). A reliable feedstock supply will be essential in maintaining stable operational costs. Further, cellulosic refineries will be required to convert biomass with potentially greater feedstock quality variability than existing corn (Zea mays L.) grain ethanol plants. Switchgrass is being developed as a biomass energy crop for the temperate regions of North America (Vogel and Mitchell, 2008). Temporal and spatial variation information across production years and fields for biomass yield and quality will be needed for establishing reliable feedstock supply areas for a cellulosic biorefinery. Information on field-scale spatial and temporal variation for biomass yield of switchgrass is becoming available (Schmer et al., 2010). Switchgrass biomass composition and theoretical ethanol production at the field-scale have thus far not been reported. Biomass conversion to transportation fuels by biochemical methods will be dependent on efficient cellulose and hemicellulose polymer hydrolysis to simple sugars and then conversion to oxygenated hydrocarbons (Himmel et al., 2007). First generation cellulosic biorefineries, using biochemical methods, will produce primarily ethanol by converting cellulose, hemicellulose, and noncell wall carbohydrates into simple sugars which are then fermented to ethanol by genetically engineered organisms (Lynd et al., 1991; Perlack et al., 2005). Lignin, an abundant phenolic polymer in cell walls, can be used for combined heat and power generation (Demirbas, 2001; Lynd and Wang, 2003; Sheehan et al., 2003). Biochemical methods involve a pretreatment to reduce cell wall recalcitrance and increase cell wall porosity, a saccharification process to hydrolyze complex polysaccharides to monosaccharides, and a fermentation process to convert monosaccharides to a biofuel (Stephanopoulos, 2007). Near-term commercialized efforts to convert lignocellulosic feedstocks to biofuels through biochemical methods will likely involve simultaneous saccharification and fermentation (SSF). Alternative conversion systems such as consolidated bioprocessing which combines the enzymatic production, hydrolysis, and fermentation process into one reactor, thus reducing capital costs and increasing biorefinery efficiency, are expected to be commercially available as well (Lynd et al., 2005). Cellulosic biomass conversion to biofuels via biochemical or thermochemical methods require more complex and ABstrAct Information on temporal and spatial variation in switchgrass (Panicum virgatum L.) biomass composition as it affects ethanol yield (L Mg–1) at a biorefinery and ethanol production (L ha–1) at the field-scale has previously not been available. Switchgrass biomass samples were collected from a regional, on-farm trial and biomass composition was determined using newly developed near-infrared reflectance spectroscopy (NIRS) prediction equations and theoretical ethanol yield (100% conversion efficiency) was calculated. Total hexose (cell wall polysaccharides and soluble sugars) concentration ranged from 342 to 398 g kg–1 while pentose (arabinose and xylose) concentration ranged from 216 to 245 g kg–1 across fields. Theoretical ethanol yield varied significantly by year and field, with 5 yr means ranging from 381 to 430 L Mg–1. Total theoretical ethanol production ranged from 1749 to 3691 L ha–1 across fields. Variability (coefficient of variation) within established switchgrass fields ranged from 1 to 4% for theoretical ethanol yield (L Mg–1) and 14 to 38% for theoretical ethanol production (L ha–1). Most fields showed a lack of spatial consistency across harvest years for theoretical ethanol yield or total theoretical ethanol production. Switchgrass biomass composition from farmer fields can be expected to have significant annual and field-to-field variation in a production region, and this variation will significantly affect ethanol or other liquid fuel yields per ton or hectare. Cellulosic biorefineries will need to consider this potential variation in biofuel yields when developing their business plans.

Cross-linking of lignin to arabinoxylan by ferulates limits in vitro rumen digestibility of grass cell walls. The effect of ferulate cross-linking on dry matter intake (DMI), milk production, and in vivo digestibility was investigated in ad libitum and restricted-intake digestion trials with lambs, and in a dairy cow performance trial using the low-ferulate sfe corn mutant. Silages of 5 inbred corn lines were fed: W23, 2 W23sfe lines (M04-4 and M04-21), B73, and B73bm3. As expected, the W23sfe silages contained fewer ferulate ether cross-links and B73bm3 silage had a lower lignin concentration than the respective genetic controls. Silages were fed as the sole ingredient to 4 lambs per silage treatment. Lambs were confined to metabolism crates and fed ad libitum for a 12-d adaptation period followed by a 5-d collection period of feed refusals and feces. Immediately following the ad libitum feeding trial, silage offered was limited to 2% of body weight. After a 2-d adaptation to restricted feeding, feed refusals and feces were collected for 5 d. Seventy Holstein cows were blocked by lactation, days in milk, body weight, and milk production and assigned to total mixed ration diets based on the 5 corn silages. Diets were fed for 28 d and data were collected on weekly DMI and milk production and composition. Fecal grab samples were collected during the last week of the lactation trial for estimation of feed digestibility using acid-insoluble ash as a marker. Silage, total mixed ration, feed refusals, and fecal samples were analyzed for crude protein, starch, neutral detergent fiber (NDF), cell wall polysaccharides, and lignin. The W23sfe silages resulted in lower DMI in the ad libitum trial than the W23 silage, but DMI did not differ in the restricted trial. No differences were observed for NDF or cell wall polysaccharide digestibility by lambs with restricted feeding, but the amount of NDF digested daily increased for lambs fed the M04-21 W23sfe silage ad libitum. Lambs were less selective against NDF and lignin when offered W23sfe silages. The B73bm3 silage did not affect DMI or digestibility of cell walls at the restricted feeding level, but total daily NDF digested was greater at ad libitum intake. Intake, milk production, and cell wall digestibility were greater for cows fed diets containing W23sfe silages than for those fed W23 silage. Although milk production was greater for the B73bm3 diet, DMI and cell wall digestibility were not altered. Cows were less selective against cell wall material when fed both W23sfe and B73bm3 silages. Reduced ferulate cross-linking in sfe corn silage is a new genetic mechanism for improving milk production.

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72 h conversion assay (CONV) with in vitro rumen microflora incubation for 72 h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r = 0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.

b Background: Knowledge about adverse symptoms over time from fiber supplementation is lacking. b Purpose: The aim of this study was to compare the severity of adverse gastrointestinal (GI) symptoms during supplementation with dietary fiber or placebo over time in adults with fecal incontinence. A secondary aim was to determine the relationship between symptom severity and emotional upset and their association with study attrition and reducing fiber dose. b Methods: Participants(N = 189; 77% female; 92% White; age, M =5 8 years,SD = 14 years) with fecal incontinence were randomly assigned to a placebo or a supplement of 16 g total dietary fiber per day from 1 of 3 sources: gum arabic, psyllium, or carboxymethylcellulose. They reported GI symptoms daily during baseline (14 days), incremental fiber dosing (6 days), and 2 segments of steady full fiber dose (32 days total). b Results: Severity of symptoms in all groups was minimal. Adjusting for study segment and day, a greater feeling of fullness in the psyllium group was the only symptom that differed from symptoms in the placebo group. The odds of having greater severity of flatus, belching, fullness, and bloating were 1.2Y2.0 times greater in the steady dose segment compared with baseline. There was a positive association between symptom severity and emotional upset. Participants with a greater feeling of fullness or bloating or higher scores for total symptom severity or emotional upset were more likely to withdraw from the study sooner or reduce fiber dose. b Conclusions: Persons with fecal incontinence experience a variety of GI symptoms over time. Symptom severity and emotional upset appear to influence fiber tolerance and study attrition. Supplements seemed well tolerated.

Switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated under ambient temperature and pressure with sulfuric acid and calcium hydroxide in separate experiments. Chemical loadings from 0 to 100 g (kg DM)−1 and durations of anaerobic storage from 0 to 180 days were investigated by way of a central composite design at two moisture contents (40% or 60% w.b.). Pretreated and untreated samples were fermented to ethanol by Saccharomyces cerevisiae D5A in the presence of a commercially available cellulase (Celluclast 1.5 L) and β-glucosidase (Novozyme 188). Xylose levels were also measured following fermentation because xylose is not metabolized by S. cerevisiae. After sulfuric acid pretreatment and anaerobic storage, conversion of cell wall glucose to ethanol for reed canarygrass ranged from 22% to 83% whereas switchgrass conversions ranged from 16% to 46%. Pretreatment duration had a positive effect on conversion but was mitigated with increased chemical loadings. Conversions after calcium hydroxide pretreatment and anaerobic storage ranged from 21% to 55% and 18% to 54% for reed canarygrass and switchgrass, respectively. The efficacy of lime pretreatment was found to be highly dependent on moisture content. Moreover, pretreatment duration was only found to be significant for reed canarygrass. Although significant levels of acetate and lactate were observed in the biomass after storage, S. cerevisiae was not found to be inhibited at a 10% solids loading.

Corn (Zea mays L.) stover, the residue left after harvest, is an abundant biomass source for pro- ducing cellulosic ethanol in the United States. Corn has been bred for increased grain yield but not for stover quality for cellulosic ethanol production. Our objective in this study was to assess the feasibility of breeding corn for grain yield and agronomic traits as well as stover quality traits for cellulosic ethanol production. Testcrosses of 223 B73 × Mo17 recombinant inbreds were evaluated at four Minnesota loca- tions in 2007. Three stover quality traits were measured: concentration of cell wall glucose in dry stover ("Glucose"); cell wall glucose released from the stover by thermochemical pretreatment and enzymatic saccharifi cation ("Glucose Release"); and concentration of lig- nin on a cell wall basis ("Lignin"). Genetic vari- ances were signifi cant for grain yield, moisture, stalk and root lodging, plant height, and all three stover quality traits. Heritabilities of the stover quality traits were 0.57 for Glucose, 0.63 for Glucose Release, and 0.68 for Lignin. Pheno- typic and genetic correlations were favorable or neutral among grain yield, agronomic traits, Glucose, Glucose Release, and Lignin. Lines selected with a multiple-trait index for grain yield, agronomic traits, and stover quality traits had improved means for each trait in the index. Current corn breeding programs should be able to incorporate stover quality for cellulosic etha- nol as a breeding objective, without having to use unadapted or exotic germplasm and with- out adversely affecting genetic gains for grain yield and agronomic traits.

In cellulosic ethanol production, the efficiency of converting maize (Zea mays L.) stover into fermentable sugars partly depends on the stover cell wall structure. Breeding for improved stover quality for cellulosic ethanol may benefit from the use of molecular markers. However, limited quantitative trait loci (QTL) studies have been published for maize stover cell wall components, and no QTL study has been published for glucose release (GLCReI) from stover by a cellulosic ethanol conversion process. Our objectives were to characterize the relationships among stover cell wall components and GLCReI, and to identify QTL with major effects, if any, influencing stover cell wall composition and GLCReI. Testcrosses of 223 intermated B73 × Mo17 recombinant inbreds and the parent lines were analyzed for cell wall composition and GLCReI after acid pretreatment and enzymatic hydrolysis. As expected, glucose (GLC), xylose (XYL), and Klason lignin (KL) composed the bulk (-72%) of the stover dry matter. Significant genetic variance and moderate heritability were observed for all traits. Genetic and phenotypic correlations among traits were generally in the favorable direction but also reflected the complexity of maize stover cell wall composition. We found 152 QTL, mostly with small effects, for GLCReI and cell wall components on both a dry matter and cell wall basis. Because no major QTL were found, methods that increase the frequency of favorable QTL alleles or that predict performance based on markers would be appropriate in marker-assisted breeding for maize stover quality for cellulosic ethanol.

Ferulate cross-linking of lignin to arabinoxylan contributes to poor cell wall degradability of grass forages. We hypothesized that reduced ferulate ester deposition will result in formation of fewer ferulate cross links and improved degradability. Objectives were to determine if the putative seedling ferulate ester (sfe) maize (Zea mays L.) mutant, selected for reduced ferulate esters in seedling leaves, (i) exhibits reduced ferulate ethers; (ii) alters morphology, yield, and cell wall concentration and composition; and (iii) changes degradability. Four near-isogenic sfe lines and backcrosses to W23 were compared with the inbred W23 in replicated fi eld trials. Leaf blade, sheath, and stem of immature (1-m plant height) and mature (silage) growth stages were analyzed for ferulate esters and ethers, cell wall concentration and composition, and in vitro rumen degradability. The sfe lines were taller and had greater internode cross-sectional area than W23. Plant yield was greater at silage maturity for sfe lines and backcross lines yielded more than sfe. Mature plant parts of sfe lines had lower ferulate ester concentrations than W23. At silage stage sfe lines had less ferulate ethers, small reductions in cell wall concentration, and shifts in composition. Immature samples were inconsistent for cell wall traits and backcross lines were generally intermediate, regardless of maturity. Cell wall degradability was higher for the sfe mutant as hypothesized. Reducing ferulate-mediated cross-linking of lignin to arabinoxylan in the cell walls of maize stover appears to be an effective strategy for improving cell wall degradability, and the sfe mutant may offer an opportunity to identify a gene controlling ferulate deposition in grasses.

Bermuda grass is an attractive candidate as a feedstock for biofuel production because over four million hectares of Bermuda grass are already grown for forage in the Southern USA. Because both rumen digestion and biochemical conversion to ethanol depend upon enzymatic conversion of the cell wall polysaccharides into fermentable sugars, it is probable that grasses bred for increased forage quality would be more amenable for ethanol production. However, it is not known how variation in rumen digestibility and cell wall/fiber components correlates with efficiency of conversion to ethanol via fermentation. The objective of this research was to determine relationships between ethanol production evaluated by simultaneous saccharification and fermentation (SSF), 72-h in vitro ruminal dry matter digestibility (IVDMD), in vitro ruminal gas production after 24 and 96 h, and biomass composition for 50 genetically diverse Bermuda grass accessions. The Bermuda grass samples were subjected to standard 72-h IVDMD and forage fiber analyses. Also, in separate labs, gas production was measured in sealed volume-calibrated vials after 24 (NNG24) and 96 h (NNG96) of in vitro fermentation by ruminal fluid; ethanol and pentose sugar productions were measured from a bench-top SSF procedure; cell wall constituents were determined by the Uppsala Dietary Fiber Method; and total nitrogen, carbon, and ash concentrations were determined by using the LECO combustion method. Ethanol production was moderately correlated with IVDMD (r = 0.55) and NNG96 (r = 0.63) but highly correlated with NNG24 (r = 0.93). Ethanol was negatively correlated with neutral detergent fiber (NDF; r = −0.53) and pentose sugars (r = −0.60), but not correlated with glucose content. Regression models indicated that NDF and cell wall pentose sugar concentrations had significant negative effects on ethanol production. Variation among entries for IVDMD was affected by variability of NDF, pentose sugar concentrations, and biomass nitrogen content. Variation in Klason lignin content had only minor negative impacts on ethanol production and IVDMD. Biochemical conversion efficiency of Bermuda grass by SSF can be best estimated by NNG24 but not by IVDMD.

Advances in alfalfa [Medicago sativa (L.) subsp. sativa] breeding, molecular genetics, and genomics have been slow because this crop is an allogamous autotetraploid (2n = 4x = 32) with complex polysomic inheritance and few genomic resources. Increasing cellulose and decreasing lignin in alfalfa stem cell walls would improve this crop as a cellulosic ethanol feedstock. We conducted genome-wide analysis of single-feature polymorphisms (SFPs) of two alfalfa genotypes (252, 1283) that differ in stem cell wall lignin and cellulose concentrations. SFP analysis was conducted using the Medicago GeneChip (Affymetrix, Santa Clara, CA) as a crossspecies platform. Analysis of GeneChip expression data fi les of alfalfa stem internodes of genotypes 252 and 1283 at two growth stages (elongating, post-elongation) revealed 10,890 SFPs in 8230 probe sets. Validation analysis by polymerase chain reaction (PCR)-sequencing of a random sample of SFPs indicated a 17% false discovery rate. Functional classifi cation and over-representation analysis showed that genes involved in photosynthesis, stress response and cell wall biosynthesis were highly enriched among SFP-harboring genes. The Medicago GeneChip is a suitable cross-species platform for detecting SFPs in tetraploid alfalfa.

Alfalfa (Medicago sativa L.) is a promising bioenergy and bioproduct feedstock because of its high yield, N-fixation capacity, potential for planting in rotation with corn (Zea mays L.), and valuable protein co-product (leaf meal). Our objective was to examine the effect of growth environment on biomass yield, cellulosic ethanol traits, and paper pulp fiber characteristics of alfalfa stems. Landscape position (summit and mild slope), season of harvest (four harvests per season), and multiple years (2005 and 2006) provided environmental variation. Alfalfa stem samples were analyzed for cell wall carbohydrate and lignin concentration. Stems were subjected to dilute acid pre-treatment, enzymatic saccharification, and pulping processes to measure relevant cellulosic ethanol and paper production traits. Landscape position was not a significant source of variation for yield or any biomass quality trait. Yields varied among harvests in 2005 (1,410–3,265 kg ha−1) and 2006 (1,610–3,795 kg ha−1). All cell wall, conversion test, and paper production traits exhibited year by harvest interactions with no clear pattern. Total carbohydrates and lignin ranged from 440 to 531 g kg−1 DM and from 113 to 161 g kg-1 DM, respectively. Release of cell wall sugars by the conversion test ranged widely (419 to 962 g kg−1 DM). Fiber traits were similarly variable with length and fine content ranging from 1.24 to 1.59 mm and from 15.2% to 21.9%, respectively. Utilizing alfalfa biomass for cellulosic ethanol and paper pulp production will involve dealing with significant feedstock quality variation due to growth environment.

We have initiated a genome-wide transcript profiling study using the model legume Medicago truncatula to identify putative genes related to cell wall biosynthesis and regulatory function in legumes. We used the GeneChip® Medicago Genome Array to compare transcript abundance in elongating versus postelongation stem internode segments of two M. truncatula accessions and two Medicago sativa (alfalfa) clones with contrasting stem cell wall concentration and composition. Hundreds of differentially expressed probe sets between elongating and postelongation stem segments showed similar patterns of gene expression in the model legume and cultivated alfalfa. Differentially expressed genes included genes with putative functions associated with primary and secondary cell wall biosynthesis and growth. Mining of public microarray data for coexpressed genes with two marker genes for secondary cell wall synthesis identified additional candidate secondary cell wall-related genes. Coexpressed genes included protein kinases, transcription factors, and unclassified groups that were not previously reported with secondary cell wall-associated genes. M. truncatula has been recognized as an excellent model plant for legume genomics. The stem tissue transcriptome analysis, described here, indicates that M. truncatula has utility as a model plant for cell wall genomics in legumes in general and shows excellent potential for translating gene discoveries to its close relative, cultivated alfalfa, in particular. The natural variation for stem cell wall traits in Medicago may offer a new tool to study an expanded repertoire of valuable agronomic traits in related species, including woody dicots in the eurosid I clade.

This study focused on the production of bio-oil from alfalfa stem material. Two alfalfa maturity stages, harvested at the early bud and full flower stages of development, were examined to evaluate the impact of variation in cell wall polysaccharide and lignin content on pyrolysis oil yields, production efficiency, and bio-oil and char quality, in terms of their use as combustion fuel and for chemicals. Findings included a lower-than-average yield of bio-oil and a higher-than-average yield of charcoal from alfalfa stems, compared to previous results for other biomass feedstocks. The bio-oil showed a decrease in oxygen content from the alfalfa stems, leading to a higher-than-average energy content in the bio-oil. Bio-oil yields were slightly higher for the more-mature alfalfa, which had higher levels of cell wall cellulose and lignin. Overall, when all the pyrolysis products were considered, energy recovery was better for the more-mature alfalfa stems.

Decreased lignin concentration or decreased ferulate cross-linking between arabinoxylans and lignin are two mechanisms to increase cellwall digestibility in plants. The objectives of this study were (i) to determine the consistency and clonal repeatability of lignin and etherifi ed ferulates across multiple harvest dates and years, (ii) to determine if the genetic correlation between lignin and etherifi ed ferulates can be altered by intensive selection, and (iii) to determine the effects of lignin and ferulates on digestibility of neutral detergent fi ber (NDF). Thirty clones each of smooth bromegrass (Bromus inermis Leyss), orchardgrass (Dactylis glomerata L.), and reed canarygrass (Phalaris arundinacea L.) were evaluated in a replicated fi eld study at four growth stages in 2004 and 2005 for NDF, lignin, esterifi ed and etherifi ed ferulates, and 24- and 96-h in vitro NDF digestibility (IVNDFD). Clonal means were generally repeatable across years and harvest dates. Divergent selection created clonal groups with differential lignin and etheri fi ed ferulates, but the positive correlation between these two traits was reduced only in smooth bromegrass. Both lignin and etherifi ed ferulates were negatively correlated with 96-h IVNDFD and these relationships were maintained as all three grasses matured. Concentration of NDF was highly correlated with etherifi ed ferulates, making it diffi cult to partition the impact of components of lignifi cation on IVNDFD.

Alfalfa (Medicago sativa L.) has been proposed as a biofuel feedstock in which the stems would be processed to produce ethanol and the leaves sold separately as a livestock feed. Our objectives were to evaluate the effects of management strategy on leaf crude protein (CP), and stem carbohydrate concentrations and yields of alfalfa germplasms differing in genetic background. Two hay-type and two biomass-type alfalfas were established at 450 plants m −2 and harvested at early bud (hay management system) and at 180 plants m −2 and harvested at green pod (biomass management system) in three environments. The biomass-type alfalfas under the biomass management had lower leaf CP, higher stem cell wall polysaccharide, and higher stem lignin concentrations, comparable leaf CP yield, and 37% greater stem cell wall polysaccharide yields compared to the hay-type alfalfas under the hay management treatment. The impact of altered stem cell wall composition and increased stem dry matter yield of a biomass-type alfalfa under the biomass system compared to a hay-type alfalfa under the hay system increased the theoretical potential ethanol yield by 99%.

Alfalfa (Medicago sativa L.) is an agronomi- cally important forage, but digestibility of stem cell wall material is low. Because the tetraploid genome of alfalfa complicates genetic dissec- tion of complex pathways, the diploid M. trun- catula Gaertn. could serve as a model for stem cell wall development in alfalfa. We compared stem morphology, chemical composition (pro- tein, soluble carbohydrates, cell wall polysac- charides, and lignin), and in vitro ruminal cell wall polysaccharide digestibility of two alfalfa clones (Regen-SY27 and 718) and four M. trun- catula inbred lines (A17, A20, DZA315.16, and R108) in a replicated growth chamber experi- ment. Stem tissue development and cell wall lignifi cation observed by light microscopy were similar between the species. While differences in stem morphology, composition, and digest- ibility were observed among the germplasms, there was overlap between the alfalfa and M. truncatula germplasms for all traits except pro- tein concentration, which was greater for the two alfalfa clones. Younger stem internodes (top third of the stem) of both species had a higher protein concentration and greater cell wall polysaccharide digestibility, and lower cell wall concentration than older internodes (bot- tom third of stem). Based on the data presented here, it appears that M. truncatula is a suitable model for stem development, composition, and digestibility of alfalfa.

Production of energy from renewable biomass resources would reduce atmospheric CO2 increase associated with fossil fuel use. The objective of this study was to evaluate the energy potential of a thermochemical conversion platform of three herbaceous biomass crops. The biomass crops tested were stems of alfalfa, a legume, and whole herbage of reed canarygrass and eastern gamagrass, cool- and warmseason grasses, respectively. Two stages of physiological development were included for the alfalfa and reed canarygrass; bud and full flower stages for the alfalfa, and vegetative and ripe seed stages for the reed canarygrass. The eastern gamagrass was fully mature, senescent material. Pyrolysis products at 600–1050 � C were characterized for gas and char yields, and non-condensable gas product composition. Gas yields ranged between 15 and 36 Nm 3 kg � 1 while char yield ranged between 5.4 and 26.7 wt% of dry initial biomass. Gas yields for alfalfa were greater than from the grasses at all temperatures ranging from 25 to 36 Nm 3 kg � 1 compared to the grasses in the combined range of 15–27 Nm 3 kg � 1 biomass. For the alfalfa stems, the more mature sample yielded more gas. For reed canarygrass, higher gas yield was obtained for the sample harvested at the vegetative stage than the more mature flowering stage. Char was greater for mature reed canarygrass than eastern gamagrass at temperatures of 900 � C or less, with the other biomass samples being intermediate. Maturity effect on alfalfa char yield was not significant. However, all samples had similarly low char residues at 1050 � C. With regard to the composition of non-condensable gas produced, the grasses had higher heating values than alfalfa; however, at 900 � C where the gas heat of combustion is maximized the calorific value of the gas was similar for all samples at about 13.6 MJ kg � 1 . This compares to about 18.4 MJ kg � 1 of the parent biomass i.e., about 75%. The activation energy for thermal decomposition, estimated from first order reaction kinetic models, did not exhibit a consistent trend with maturity, but mean activation energy was lower for alfalfa (2837 kJ mol � 1 ) than the grasses (3427 and 3419 kJ mol � 1 for reed canarygrass and eastern gamagrass, respectively). The effect of maturity on the pyrolysis

Seasonal time of switchgrass (Panicum virgatum L.) harvest affects yield and biofuel quality and balancing these two components may vary depending on conversion system. A field study compared fall and spring harvest measuring biomass yield, element concentration, carbohydrate characterization, and total synthetic gas production as indicators of biofuel quality for direct combustion, ethanol production, and gasification systems for generation of energy. Switchgrass yields decreased almost 40% (from about 7–4.4 Mg ha 21 ) in winters with above average snowfall when harvest was delayed over winter until spring. The moisture concentration also decreased (from about 350– 70 g kg 21 ) only reaching low enough levels for safe storage by spring. About 10% of the yield reduction during winter resulted from decreases in tiller mass; however, almost 90% of the yield reduction was due to an increase in biomass left behind by the baler. Mineral element concentrations generally decreased with the delay in harvest until spring. Energy yield from gasification did not decrease on a unit biomass basis, whereas ethanol production was variable depending on the assessment method. When expressed on a unit area basis, energy yield decreased. Biofuel conversion systems may determine harvest timing. For direct combustion, the reduced mineral concentrations in spring-harvested biomass are desirable. For ethanol fermentation and gasification systems, however, lignocellulose yield may be more important. On conservations lands, the wildlife cover provided by switchgrass over the winter may increase the desirability of spring harvest along with the higher biofuel quality.

Alfalfa stems, reed canarygrass, and switchgrass; perennial herbaceous species that have potential as biomass energy crops in temperate regions; were evaluated for their bioconversion potential as energy crops. Each forage species was harvested at two or three maturity stages and analyzed for carbohydrates, lignin, protein, lipid, organic acids, and mineral composition. The biomass samples were also evaluated for sugar yields following pretreatment with dilute sulfuric followed by enzymatic saccharification using a commercial cellulase preparation. Total carbohydrate content of the plants varied from 518 to 655 g kg � 1 dry matter (DM) and cellulose concentration from 209 to 322 g kg � 1 DM. Carbohydrate and lignin contents were lower for samples from early maturity samples compared to samples from late maturity harvests. Several important trends were observed in regards to the efficiency of sugar recovery following treatments with dilute acid and cellulase. First, a significant amount of the available carbohydrates were in the form of soluble sugars and storage carbohydrates (4.3–16.3% wt/wt). Recovery of soluble sugars following dilute acid pretreatment was problematic, especially that of fructose. Fructose was found to be extremely labile to the dilute acid pretreatments. Second, the efficiency at which available glucose was recovered was inversely correlated to maturity and lignin content. However, total glucose yields were higher for the later maturities because of higher cellulose contents compared to the earlier maturity samples. Finally, cell wall polysaccharides, as determined by the widely applied detergent fiber system were found to be inaccurate. The detergent fiber method consistently overestimated cellulose and hemicellulose and underestimated lignin by substantial amounts. Published by Elsevier Ltd.

Poor cell wall digestibility of alfalfa (Medicago sativa L.) stems limitsavailableenergytoruminants.Thisstudycomparedalfalfaclones identified as either low or high rapid (16 h), or low or high potential (96 h) stem in vitro neutral detergent fiber digestibility (IVNDFD) for stem detergent fiber, cell wall, and morphology traits. Clones were established in replicated field plots at two sites in Minnesota during 2001 and harvested twice (primary spring growth and first summer regrowth) in the two following years. Presence of flowers, stem length, internode number, mean internode length, and number of elongating internodes was determined on 10 stems from each plot at every harvest. Herbage was dried and the stem fraction was analyzed for 16- and 96-h IVNDFD, detergent fiber components, and cell wall concentration and composition (Klason lignin, individual neutral sugars, and total uronic acids). Stem neutral detergent fiber (NDF) and cell wall concentration were lower for the high rapid than low rapid IVNDFD clones, but the high and low potential IVNDFD clonal groups did not differ. Lignin and pectin concentrations of the cell wall were lower and higher, respectively, for both high IVNDFD groups than their corresponding low groups. The low rapid and high potential IVNDFD groups had longer stem and internode lengths than their corresponding groups.

We determined the effect of N fertilization on dry matter (DM) yield, predicted milk yield, and forage quality of fresh (green chopped) and ensiled forage of two brown midrib (BMR) hybrids, a leafy hybrid, and a conventional silage hybrid. Increasing N rates from 0 to 200 kg ha -1 increased corn grain, stover and whole plant DM yield and milk yield. The forage yield and quality response to N was similar for all hybrids. Nitrogen fertilization increased forage crude protein (CP) concentration but had little effect on other forage quality components. BMR hybrids, F377 and F657, had the lowest grain, stover and whole plant yield but had the highest digestibility and predicted milk yield Mg -1 of forage. Predicted milk yield ha -1 was similar for the BMR, leafy and conventional hybrids. Ensiling reduced starch concentration compared with green chopped forage, but effects on other forage quality variables were less consistent. Hybrid and N fertilization affects were similar for green chopped and ensiled corn forage.

During soybean [Glycine max (L.) Merrill] embryo development, cell wall polysaccharides (CWPs) derived from UDP-glucuronic acid (UDP-GlcA) (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25–45 days after flowering (daf). At embryo maturity, CWPs derived from UDP-GlcA accounted for 39% of total CWPs. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo-inositol oxidation (MIO) pathways to UDP-GlcA biosynthesis, UDP-glucose (UDP-Glc) dehydrogenase (UDP-Glc DH, EC 1.1.1.22) and UDP-glucuronic acid pyrophosphorylase (UDP-GlcA PPase, EC 2.7.7.44) activities, respectively, were measured in desalted extracts of developing embryos. UDP-Glc DH and UDP-GlcA PPase activities, expressed on a per seed basis, increased 3.5- and 3.9-fold, respectively, during the period of 25–45 daf. However, UDP-GlcA PPase activity was 35–50-fold greater than UDP-Glc DH activity. The soybean UDP-sugar pyrophosphorylase gene (USP1), a homolog of pea USP, and a candidate gene for UDP-GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with glucuronic acid 1-phosphate (GlcA-1-P), glucose 1-phosphate (Glc-1-P) and galactose 1-phosphate (Gal-1-P), but low activity with mannose 1-phosphate (Man-1-P), N-acetylglucosamine 1-phosphate and Glc-6-P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA-1-P (Km= 0.14 ± 0.02 mM) than for Glc-1-P (Km= 0.23 ± 0.02 mM). Semiquantitative RT-PCR was used to measure transcript levels of the UDP-glucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as UDP-GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.

Grass degradability declines as cell wall and lignin concentrations increase during maturation. The role of tissue development and lignification in decline of stem degradability was examined in maize (Zea mays L.) internodes sampled at 10 stages of growth from early elongation through plant physiological maturity. The fourth elongated internode above ground level was collected from three maize hybrids grown in a 2-yr, replicated field trial at St. Paul, MN. Internode cross-sections and ground samples were incubated in vitro for 24- and 96-h with rumen microbes. Tissue degradation was examined by light microscopy and degradability of cell wall polysaccharide components from ground internodes was determined. All tissues in elongating internodes were completely degradable except for protoxylem vessels, which was the only lignified tissue. After elongation, degradability of all tissues declined markedly except for phloem, which never lignified. Tissues with thick, lignified secondary walls (sclerenchyma and rindregion parenchyma) required longer incubation times for observable degradation. Cell wall polysaccharide components were highly degradable in immature internodes, but degradability declined after elongation and reached a minimum by Sampling Date 8, with glucose and xylose residues having the greatest reductions in degradability. Cell wall polysaccharide degradation was related to lignin concentration and ferulate cross-linking in a complex manner.

Livestock performance can be improved by increasing the digestibility of feeds, one of the key elements of nutritional quality. Digestibility of feeds can be improved by breeding forage crops with modified cell-wall structure, increasing the potential availability of energy from the cell wall to rumen microbes and livestock. The objectives of this research were to identify interrelationships among lignin and phenolic components of the fibre fraction of three perennial grasses and to determine their influence on in vitro fibre digestibility. Differences in etherified and esterified ferulate and esterified p-coumarate among clones of three perennial grasses were generally repeatable across harvests. The concentration of neutral detergent fibre (NDF) and Klason lignin within the NDF fraction were the factors most limiting to 24-h in vitro digestibility, with NDF being the most important. Klason lignin and etherified ferulate were the factors most limiting to 96-h in vitro digestibility for all three species. Due to its positive correlation with NDF, selection for low etherified ferulate should be avoided in smooth bromegrass (Bromus inermis Leyss) and cockfsoot (Dactylis glomerata L.). It should be possible to select and breed for low concentrations of lignin to improve digestibility without decreasing NDF in these two species. However, in reed canarygrass (Phalaris arundinacea L.), both lignin and etherified ferulate were positively correlated with NDF, indicating that selection for increased digestibility should be based directly on some measure of in vitro digestibility to avoid the fitness problems associated with reduced NDF.

Seed cell wall polysaccharides (CWPs) represent a significant portion of seed dry matter (DM) in soybean [Glycine max (L.) Merr.]. Quantitative trait locus (QTL) analysis is a step toward identifying genes controlling CWP concentration and composition of soybean seed. The objectives of this study were to identify QTLs associated with CWP variability and investigate the relationships between seed CWP, protein, and oil content. Whole soybean seed from 'Minsoy', 'Archer', and 108 Minsoy × Archer recombinant inbred (RI) lines were analyzed for CWP using the Uppsala total dietary fiber method. For a random subsample of 73 RI lines, embryos (seed with the seed coats removed) were analyzed. Three QTLs were observed on Linkage Groups U3 (ISU A2), U7 (ISU Al), and U24 (ISU K) that represented most of the variability in CWP content expressed on a DM basis in both whole seed and embryos. A QTL for fucose was detected on U3. Linkage Group U7 contained multiple QTLs mapping at the same location for galactose, the arabinose-to-galactose ratio (Ara/ Gal), pectin, and CWP content. A QTL for arabinose was on U24. These same chromosomal regions also exhibited significant QTLs when the monosaccharide data were expressed on a CWP basis, suggesting that most of the variation in CWP was controlled by changes in the incorporation of galactose, arabinose, and fucose into CWP as total CWP concentration increased. Negative correlations of protein with oil, and the sum of protein and oil with most monosaccharides, pectin, and CWP were present within the RI population, indicating that decreasing seed CWP content will improve seed quality.

Identification of superior biomass feedstocks for conversion to ethanol requires accurate, rapid, and inexpensive measurement of cell wall polysaccharide and lignin concentrations. A study was undertaken to determine if cell wall characteristics measured with the Uppsala dietary fiber method could be accurately predicted from detergent fiber data. Spring and summer growth alfalfa (Medicago sativa L.) stems were harvested from two germplasm sources for 2-years. Samples were analyzed for neutral sugars, uronic acids, and Klason lignin using the Uppsala dietary fiber method, and neutral detergent fiber, acid detergent fiber, and acid detergent lignin by sequential detergent analysis. Cell wall, hemicellulose, cellulose, and lignin concentrations derived from these two methods were positively correlated, but detergent estimates of cell wall and lignin concentration were lower and hemicellulose and cellulose concentrations were higher than for the Uppsala method. These patterns were attributed to solubility of pectin in neutral detergent solution and poor lignin recovery in acid detergent lignin. Regression equations for prediction of cell wall characteristics using detergent results were significant and accounted for 35–92% of the variation in the data. Significant deviations between predicted and observed cell wall results were observed when the equations were tested using an independent sample set, although the deviation was very small for cellulose concentration. It does not appear that most cell wall characteristics of alfalfa stems obtained using the Uppsala dietary fiber method can be accurately predicted using detergent fiber results; however, cellulose concentration might be predicted with acceptable accuracy.