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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

Corn stover ethanol yield as affected by grain yield, Bt trait, and environment

Pavani Tumbalam; Kurt D. Thelen; Andrew Adkins; Bruce Dale; Venkatesh Balan; Christa Gunawan; Juan Gao

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2016

Literature values for glucose release from corn stover are highly variable which would likely result in tremendous variability in bio-refinery ethanol yield from corn stover feedstock. A relatively recent change in United States corn genetics is the inclusion of the Bacillus thuringiensis (Bt) trait, which now accounts for three-fourths of all US planted corn acreage. The objective of this study was to evaluate the effect of corn grain yield, inclusion of the Bt trait, and location environment on corn stover quality for subsequent ethanol conversion. Two hybrid pairs (each having a Bt and non-Bt near-isoline) were analyzed giving a total of 4 hybrids. In 2010 and 2011, field plots were located in Michigan at four lat- itudinal differing locations in four replicated plots at each location. Stover composition and enzymatic digestibility was analyzed and estimated ethanol yield (g g 1) was calculated based on hydrolyzable glucan and xylan levels. Analysis showed that there were no significant differences in total glucose or xylose levels nor in enzymatically hydrolyzable glucan and xylan concentrations between Bt corn stover and the non-Bt stover isolines. Regression analyses between corn grain yield (Mg ha 1) and corn stover ethanol yield (g g 1) showed an inverse relationship indicative of a photosynthate source-sink rela- tionship. Nevertheless, the quantity of stover produced was found to be more critical than the quality of stover produced in maximizing potential stover ethanol yield on a land area basis.

Designer lignins: Harnessing the plasticity of lignification

Yaseen Mottiar; Ruben Vanholme; Wout Boerjan; John Ralph; Shawn D. Mansfield

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2016

Lignin is a complex polyphenolic constituent of plant secondary cell walls. Inspired largely by the recalcitrance of lignin to biomass processing, plant engineering efforts have routinely sought to alter lignin quantity, composition, and structure by exploiting the inherent plasticity of lignin biosynthesis. More recently, researchers are attempting to strategically design plants for increased degradability by incorporating monomers that lead to a lower degree of polymerisation, reduced hydrophobicity, fewer bonds to other cell wall constituents, or novel chemically labile linkages in the polymer backbone. In addition, the incorporation of value-added structures could help valorise lignin. Designer lignins may satisfy the biological requirement for lignification in plants while improving the overall efficiency of biomass utilisation.

Investment risks in bioenergy crops

T. Skevas; S.M. Swinton; S. Tanner; G. Sanford; K.D. Thelen

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2016

Nanoscale structure of biomass

Shi-You Ding

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2016

Plant biomass is a renewable source that can be processed to produce biofuels and biomaterials. The plant cell walls are the major material in biomass. Depending on plant species and the time of harvest, biomass varies in its anatomical structure and chemical composition. This chapter summarizes general structure of the plant cell walls in different plant tissues and plant species, and updates are given from new findings in in situ imaging at nanoscale resolution and real-time changes during biomass deconstruction processes. The physicochemical properties of biomass that affect the efficiency of thermochemical pretreatment and enzymatic hydrolysis are also discussed.

Xenorhabdus bovienii s  train diversity impacts coevolution and symbiotic maintenance with Steinernema  spp. nematode hosts

Kristen E. Murfin; Ming-Min Lee; Jonathan L. Klassen; Bradon R. McDonald; Bret Larget; Steven Forst; Patricia Stock; Cameron R. Currie; Heidi Goodrich-Blair

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2015

A cardiolipin-deficient mutant of Rhodobacter sphaeroides  has an altered cell shape and is impaired in biofilm formation

Ti-Yu Lin; Thiago M.A. Santos; Wayne S. Kontur; Timothy J. Donohue; Douglas B. Weibel

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2015

Cell shape has been suggested to play an important role in regulating bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase mutant (Deltacls) of the rod-shaped bacterium Rhodobacter sphaeroides-in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%-produces ellipsoidal-shaped cells that are impaired in forming biofilms. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 mug/mL) of the small molecule MreB inhibitor, S-(3,4-dichlorobenzyl)isothiourea (A22), produced ellipsoidal-shaped cells that had no obvious growth defect, yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of this bacterium to adapt to its environment. IMPORTANCE: Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid, cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in regulating R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.

A consistent positive association between landscape simplification and insecticide use across the Midwestern US from 1997 through 2012

Timothy D. Meehan; Claudio Gratton

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2015

During 2007, counties across the Midwestern US with relatively high levels of landscape simplification (i.e., widespread replacement of seminatural habitats with cultivated crops) had relatively high crop-pest abundances which, in turn, were associated with relatively high insecticide application. These results suggested a positive relationship between landscape simplification and insecticide use, mediated by landscape effects on crop pests or their natural enemies. A follow-up study, in the same region but using different statistical methods, explored the relationship between landscape simplification and insecticide use between 1987 and 2007, and concluded that the relationship varied substantially in sign and strength across years. Here, we explore this relationship from 1997 through 2012, using a single dataset and two different analytical approaches. We demonstrate that, when using ordinary least squares (OLS) regression, the relationship between landscape simplification and insecticide use is, indeed, quite variable over time. However, the residuals from OLS models show strong spatial autocorrelation, indicating spatial structure in the data not accounted for by explanatory variables, and violating a standard assumption of OLS. When modeled using spatial regression techniques, relationships between landscape simplification and insecticide use were consistently positive between 1997 and 2012, and model fits were dramatically improved. We argue that spatial regression methods are more appropriate for these data, and conclude that there remains compelling correlative support for a link between landscape simplification and insecticide use in the Midwestern US. We discuss the limitations of inference from this and related studies, and suggest improved data collection campaigns for better understanding links between landscape structure, crop-pest pressure, and pest-management practices.

A lignocellulosic ethanol strategy via nonenzymatic sugar production: process synthesis and analysis

Jeehoon Han; Jeremy S. Luterbacher; David M. Alonso; James A. Dumesic; Christos T. Maravelias

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2015

The work develops a strategy for the production of ethanol from lignocellulosic biomass. In this strategy, the cellulose and hemicellulose fractions are simultaneously converted to sugars using a gamma-valerolactone (GVL) solvent containing a dilute acid catalyst. To effectively recover GVL for reuse as solvent and biomass-derived lignin for heat and power generation, separation subsystems, including a novel CO2-based extraction for the separation of sugars from GVL, lignin and humins have been designed. The sugars are co-fermented by yeast to produce ethanol. Furthermore, heat integration to reduce utility requirements is performed. It is shown that this strategy leads to high ethanol yields and the total energy requirements could be satisfied by burning the lignin. The integrated strategy using corn stover feedstock leads to a minimum selling price of $5 per gallon of gasoline equivalent, which suggests that it is a promising alternative to current biofuels production approaches.

A reassessment of the contribution of soybean biological nitrogen fixation to reactive N in the environment

Ilya Gelfand; Philip Robertson

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2015

The expansion of soybean (Glycine max (L.) Merr) acreage, increasing yields, and recent nitrogen (N) fertilization recommendations could have a major effect on the contribution of biological N fixation (BNF) in soybeans to reactive nitrogen (Nr) in the environment. We used 15N natural abundance to separate fixed N into grain, aboveground vegetative biomass, and roots along a 9-point N-fertilizer gradient to ask: 1) is the belowground BNF contribution sufficiently different from aboveground to affect regional estimates of soybean Nr production based on harvested biomass, and 2) how does N fertilizer affect soybean yield and BNF’s contribution to different tissues? The contribution of root and vegetative biomass to overall plant BNF was five times lower than that for grain. Including this difference in BNF extrapolations translates to 3.5 ± 0.5 Tg Nr yr-1 for total US soybean production, *37 % lower than earlier estimates that did not differentiate tissue source. Production of Nr ranged between 35 ± 11 and 73 ± 5 g Nr kg-1 grain and was affected by both fertilization and irrigation. In all cases N credits to the next rotational crop were minor. N-fertilization at even very low levels (17–50 kg N ha-1) did not affect yield, but grain N content increased with fertilizer level. The percent BNF contributed to plant N decreased linearly with increasing fertilization, in grain from 49 ± 8 % in unfertilized plots to a plateau of 16 ± 6 % at fertilization C85 kg N ha-1; in aboveground vegetative biomass from 77 ± 4 % to aplateauof11 ± 11 %at146 kg Nha-1;andinroots from 88±12% to a plateau of 41±6% at 146 kg N ha-1. The average whole-plant BNF con- tribution decreased from *84 % in unfertilized plots to a plateau of *34 % at fertilization rates greater than 84 kg ha-1. Results underscore the unnecessary expense and environmental burden of adding N fertilizer to modern soybean varieties, and provide a refined lower estimate for the contribution of soybean N fixation to the US and global Nr budgets of 3.5 and 10.4 Tg Nr yr-1, respectively.

A unique ecological niche fosters hybridization of oak-tree and vineyard isolates of Saccharomyces cerevisiae 

Katie J. Clowers; Jessica L. Will; Audrey P. Gasch

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2015

Differential adaptation to distinct niches can restrict gene flow and promote population differentiation within a species. However, in some cases the distinction between niches can collapse, forming a hybrid niche with features of both environments. We previously reported that distinctions between vineyards and oak soil present an ecological barrier that restricts gene flow between lineages of Saccharomyces cerevisiae. Vineyard isolates are tolerant to stresses associated with grapes while North American oak strains are particularly tolerant to freeze-thaw cycles. Here, we report the isolation of S. cerevisiae strains from Wisconsin cherry trees, which display features common to vineyards (e.g. high sugar concentrations) and frequent freeze-thaw cycles. Genome sequencing revealed that the isolated strains are highly heterozygous and represent recent hybrids of the oak x vineyard lineages. We found that the hybrid strains are phenotypically similar to vineyard strains for some traits, but are more similar to oak strains for other traits. The cherry strains were exceptionally good at growing in cherry juice, raising the possibility that they have adapted to this niche. We performed transcriptome profiling in cherry, oak and vineyard strains and show that the cherry-tree hybrids display vineyard-like or oak-like expression, depending on the gene sets, and in some cases, the expression patterns linked back to shared stress tolerances. Allele-specific expression in these natural hybrids suggested concerted cis-regulatory evolution at sets of functionally regulated genes. Our results raise the possibility that hybridization of the two lineages provides a genetic solution to the thriving in this unique niche.

Active site and laminarin binding in glycoside hydrolase family 55

Christopher M. Bianchetti; Taichi E. Takasuka; Sam Deutsch; Hannah S. Udell; Eric J. Yik; Lai F. Bergeman; Brian G. Fox

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2015

The Carbohydrate Active Enzyme (CaZY) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-beta -1,3-glucanases. The founding structure of the GH55 is PcLam55A from the white-rot fungus Phanaerochaete chrysosporium (Ishida, T., et al. (2009) J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies implicate Glu502 as the catalytic acid (as proposed earlier for Glu663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the non-redundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities, and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate GH phylogenetic space for functional properties.

All biomass is local: The cost, volume produced, and global warming impact of cellulosic biofuels depend strongly on logistics and local conditions

Seungdo Kim; Bruce E. Dale

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2015

Current models of cellulosic biofuel systems require that the delivered price of the cellulosic biomass feedstock be kept low. Thus the predicted biorefinery size is relatively small, limiting potential economies of scale. However, it is actually the ultimate selling price of the biofuel that largely determines market penetration. We relaxed the constraint of low delivered feedstock price and explored the resulting effects on biofuel price, biofuel volume produced, and global warming impact (GWI). Feedstock price greatly affects the feedstock supply chains that may develop. Increased feedstock price does not affect the final ethanol selling price very much, but higher feedstock prices greatly increase the amount of ethanol produced. Farmers will supply much more cellulosic biomass at higher feedstock prices, leading to shorter transportation distances with reduced transportation costs and enabling larger biorefineries with improved economies of scale, thereby reducing the ethanol selling price. The cellulosic feedstock supply chain systems were studied as a function of feedstock prices by determining potential feedstock supply clusters and the maximum capacity of cellulosic biorefineries across the United States. Supply clusters were determined by minimizing costs associated with ethanol production. The analysis is based on county-level cellulosic feedstock production data projected in the US Billion-Ton Update report. Each biomass supply cluster is unique in terms of local and regional characteristics (e.g. area, feedstock types), biorefinery capacity, ethanol selling price, and GWI. Very large-scale biorefineries (≥20 000 dry Mg day−1) may be feasible in some regions. © 2015 Society of Chemical Industry and John Wiley & Sons, Ltd

An expanded maize gene expression atlas based on RNA-sequencing and its use to explore root development

Scott C. Stelpflug; Rajandeep S. Sekhon; Brieanne Vaillancourt; Candice N. Hirsch; Robin Buell; Natalia de Leon; Shawn M. Kaeppler

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2015

Comprehensive and systematic transcriptome profiling provides valuable insight into biological and developmental processes that occur throughout the life cycle of a plant. We have enhanced our previously published microarray-based gene atlas of maize (Zea mays L.) inbred B73 to now include 79 distinct replicated samples that have been interrogated using RNA sequencing (RNAseq). The current version of the atlas includes 50 original arraybased gene atlas samples, a time-course of 12 stalk and leaf samples postflowering, and an additional set of 17 samples from the maize seedling and adult root system. The entire dataset contains 4.6 billion mapped reads, with an average of 20.5 million mapped reads per biological replicate, allowing for detection of genes with lower transcript abundance. As the new root samples represent key additions to the previously examined tissues, we highlight insights into the root transcriptome, which is represented by 28,894 (73.2%) annotated genes in maize. Additionally, we observed remarkable expression differences across both the longitudinal (four zones) and radial gradients (cortical parenchyma and stele) of the primary root supported by fourfold differential expression of 9353 and 4728 genes, respectively. Among the latter were 1110 genes that encode transcription factors, some of which are orthologs of previously characterized transcription factors known to regulate root development in Arabidopsis thaliana (L.) Heynh., while most are novel, and represent attractive targets for reverse genetics approaches to determine their roles in this important organ. This comprehensive transcriptome dataset is a powerful tool toward understanding maize development, physiology, and phenotypic diversity.

An integrated approach to reconstructing genome-scale transcriptional regulatory networks

Saheed Imam; Daniel R. Noguera; Timothy J. Donohue

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2015

Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

Analysis of a modern hybrid and an ancient sugarcane Implicates a complex interplay of factors in affecting recalcitrance to cellulosic ethanol production

Viviane Guzzo de C. Poelking; Andrea Giordano; Maria Esther Ricci-Silva; Thomas Christophe Williams; Diego Alves Pecanha; Marília Contin Ventrella; Jorge Rencoret; John Ralph; Márcio Henrique P. Barbosa; Marcelo Loureiro

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2015

Abundant evidence exists to support a role for lignin as an important element in biomass recalcitrance. However, several independent studies have also shown that factors apart from lignin are also relevant and overall, the relative importance of different recalcitrance traits remains in dispute. In this study we used two genetically distant sugarcane genotypes, and performed a correlational study with the variation in anatomical parameters, cell wall composition, and recalcitrance factors between these genotypes. In addition we also tracked alterations in these characteristics in internodes at different stages of development. Significant differences in the development of the culm between the genotypes were associated with clear differential distributions of lignin content and composition that were not correlated with saccharification and fermentation yield. Given the strong influence of the environment on lignin content and composition, we hypothesized that sampling within a single plant could allow us to more easily interpret recalcitrance and changes in lignin biosynthesis than analysing variations between different genotypes with extensive changes in plant morphology and culm anatomy. The syringyl/guaiacyl (S/G) ratio was higher in the oldest internode of the modern genotype, but S/G ratio was not correlated with enzymatic hydrolysis yield nor fermentation efficiency. Curiously we observed a strong positive correlation between ferulate ester level and cellulose conversion efficiency. Together, these data support the hypothesis that biomass enzymatic hydrolysis recalcitrance is governed by a quantitative heritage rather than a single trait.

Applications of constraint-based models for biochemical production

Cameron Cotten; Jennifer L. Reed

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2015

Biofuels are metabolic products, and knowledge of how metabolism operates is critical to understanding and improving biofuel production by microorganisms. Constraint-based metabolic modeling has been an important technique to broaden and deepen our knowledge of microbial metabolism and regulation. Genome-scale metabolic models enable global analysis of microbial metabolism by considering all metabolic reactions simultaneously. Genome-scale metabolic reconstructions are comprehensive listings of all the reactions, compounds, and genes that are involved in cellular metabolism for a particular organism. Constraint-based modeling methods use the information in metabolic reconstructions to predict intracellular fluxes and design strains for chemical and biofuel production. Recently, constraint-based modeling has been successful in designing a number of chemical production strains.

Bioenergy supply and environmental impacts on cropland: Insights from multi-market forecasts in a Great Lakes subregional bioeconomic model

Aklesso Egbendewe-Mondzozo; Scott M. Swinton; Shujiang Kang; Wilfred M. Post; Julian C. Binfield; Wyatt Thompson

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2015

Using subregional models of crop production choices in central Wisconsin and southwest Michigan, we predict biomass production, land use, and environmental impacts with details that are unavailable from national scale models. When biomass prices are raised exogenously, we find that the subregional models overestimate the supply, the land use, and the beneficial environmental aspects of perennial biomass crops. Multi-market price feedbacks tied to realistic policy parameters predict high threshold absolute prices for biomass to enter production and less environmental benefice from perennial biomass crops production. Also, regional specialization of biomass production in areas with lower food crop yields is observed.

Biomass potential of switchgrass and miscanthus on the USA’s marginal lands

Varaprasad Bandaru; César Izaurralde; Kaiguang Zhao

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2015

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