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GLBRC's Publications

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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

2D NMR characterization of wheat straw residual lignin after dilute acid pretreatment with different severities

Anders Jensen; Yohanna Cabrera; Chia-Wen Hsieh; John Nielsen; John Ralph; Claus Felby

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2017

The chemical characteristics of wheat straw lignin pretreated under dilute acid conditions were compared. After pretreatment, the lignin content of the solid residue increased as temperature increased (from 160°C to 190°C) and with the amount of acid added (0%, 0.25%, or 1% H2SO4). Pretreatment at 190°C with increasing concentrations of acid catalyst led to a decrease in glucan content, whereas the glucan content remained almost constant at 160°C pretreatment regardless of the acid concentration. The xylan content decreased in proportion with increased acid concentration and pretreatment temperature. The residual lignins were characterized by solution-state, two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and size-exclusion chromatography (SEC). Results showed that more ether bonds were cleaved with increased pretreatment temperature and lower pH, whereas the levels of carbon-carbon bonded structures (e.g. phenylcoumaran and resinol units) were hardly affected. With a pretreatment of 160°C and 1% H2SO4, the majority of the β-O-4 bonds were cleaved. In addition, lignin depolymerization was more evident than repolymerization at higher pretreatment temperatures and lower pH. Documenting lignin structural changes as a function of pretreatment parameters provides a tool for biorefineries to gain flexibility in processing parameters with full control over the final properties of the products.

A co-solvent hydrolysis strategy for the production of biofuels: process synthesis and technoeconomic analysis

Wangyun Won; Ali H. Motagamwala; James A. Dumesic; Christos T. Maravelias

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2017

We develop an integrated strategy for the production of ethanol from lignocellulosic biomass. Cellulose and hemicellulose fractions are first hydrolyzed into sugars using a mixture of γ-valerolactone (GVL), water, and toluene as a solvent containing dilute sulfuric acid as a catalyst, and the sugars are then co-fermented into ethanol over engineered yeast strains. Separation subsystems are designed to effectively recover GVL and toluene for reuse in biomass hydrolysis, and to recover lignin and humins for heat and power generation. We also develop an alternative process, in which we recover sugars and GVL from the residual biomass. To minimize utility requirements, we conduct heat integration, which allows us to meet all heating requirement using biomass residues. Finally, we perform a range of system-level analyses to identify the major cost and technological drivers. The proposed strategy is shown to be cost-competitive with other strategies.

A superstructure-based framework for bio-separation network synthesis

WenZhao Wu; Kirti Yenkie; Christos T. Maravelias

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2017

Modern biotechnologies enable the production of chemicals using engineered microorganisms. However, the cost of downstream recovery and purification steps is high, which means that the feasibility of bio-based chemicals production depends heavily on the synthesis of cost-effective separation networks. To this end, we develop a superstructure-based framework for bio-separation network synthesis. Based on general separation principles and insights obtained from industrial processes for specific products, we first identify four separation stages: cell treatment, product phase isolation, concentration and purification, and refinement. For each stage, we systematically implement a set of connectivity rules to develop stage-superstructures, all of which are then integrated to generate a general superstructure that accounts for all types of chemicals that can be produced using microorganisms. We further develop a superstructure reduction method to solve specific instances, based on product attributes, technology availability, case-specific considerations, and final product stream specifications. A general optimization model, including short-cut models for all technologies, is formulated. The proposed framework enables preliminary synthesis and analysis of bio-separation networks, and thus estimation of separation costs.

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production

Eric Poliner; Jane A. Pulman; Krzysztof Zienkiewicz; Kevin Childs; Christoph Benning; Eva M. Farre

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2017

Nannochloropsis oceanica is an oleaginous microalga rich in omega3long-chain polyunsaturated fatty acids (LC-PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC-PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC-PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779 we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC-OX and pNOC-stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC-PUFAs, with the overexpression of Delta12 and Delta5 FAD encoding sequences leading to an increase in the final omega3 product, EPA. This article is protected by copyright. All rights reserved.

Biochemical transformation of lignin for deriving valued commodities from lignocellulose

Daniel L. Gall; J. Ralph; Timothy J. Donohue; Daniel R. Noguera

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2017

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin’s renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise to a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.

Biomass and biofuel crop effects on biodiversity and ecosystem services in the North Central US

Douglas A. Landis; Claudio Gratton; Randall D. Jackson; Katherine L. Gross; David S. Duncan; Chao Liang; Timothy D. Meehan; Bruce A. Robertson; Thomas M. Schmidt; Karen A. Stahlheber; James M. Tiedje; Benjamin P. Werling

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2017

Biomass cropping systems have the potential to alter the ecosystem services provided by agricultural landscapes. Depending on crop type and management, strategic incorporation of biomass cropping systems into existing agricultural landscapes could enhance a range of ecosystem services while mitigating some disservices. Here, we review the approaches and findings of eight years of research into the potential effects of a range of biomass cropping systems on ecosystem services in the North Central US. Our research was framed by an initial assessment of the abundance and distribution of multiple taxa (i.e., biodiversity) within candidate biomass cropping systems. The processes underpinning important ecosystem services in each system were then measured or modeled, related to biodiversity metrics, and used to explore the influence of management scenarios on biodiversity and ecosystem processes. We also used these data and models to develop a decision support system that allows stakeholders to consider tradeoffs and synergies under alternative landscape composition, configuration, and agronomic management. Perennial grass cropping systems provided the greatest potential to promote multiple ecosystem services. More diverse perennial grasslands that include forbs have the potential to increase pest suppression and pollination, decrease greenhouse gas emissions, and enhance grassland bird communities, but likely at the expense of biomass yield. Providing stakeholders and policymakers with information about the expected mix of ecosystem services supported by different biomass feedstock cropping systems in advance of their adoption offers the potential for informed choices to guide the implementation and management of future biomass-producing landscapes.

Brachypodium as an experimental system for the study of stem parenchyma biology in grasses

Jacob Krüger Jensen; Curtis Gene Wilkerson

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2017

Stem parenchyma is a major cell type that serves key metabolic functions for the plant especially in large grasses, such as sugarcane and sweet sorghum, where it serves to store sucrose or other products of photosynthesis. It is therefore desirable to understand the metabolism of this cell type as well as the mechanisms by which it provides its function for the rest of the plant. Ultimately, this information can be used to selectively manipulate this cell type in a controlled manner to achieve crop improvement. In this study, we show that Brachypodium distachyon is a useful model system for stem pith parenchyma biology. Brachypodium can be grown under condition where it resembles the growth patterns of important crops in that it produces large amounts of stem material with the lower leaves senescing and with significant stores of photosynthate located in the stem parenchyma cell types. We further characterize stem plastid morphology as a function of tissue types, as this organelle is central for a number of metabolic pathways, and quantify gene expression for the four main classes of starch biosynthetic genes. Notably, we find several of these genes differentially regulated between stem and leaf. These studies show, consistent with other grasses, that the stem functions as a specialized storage compartment in Brachypodium.

Bumble bee colony growth and reproduction depend on local flower dominance and natural habitat area in the surrounding landscape

Brian J. Spiesman; Ashley Bennett; Rufus Isaacs; Claudio Gratton

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2017

Conservation measures for bees often focus on increasing the diversity and abundance of floral resources. But it has not been clear if observed benefits of floral enhancements result from greater population growth, which is critical for the long-term success of conservation, or from mobile foragers aggregating in high-resource locations. Experimental evidence is only beginning to emerge in favor of the former mechanism and it is not well-established how different aspects of floral resources affect population growth. For example, bumble bee colonies may benefit from greater overall floral abundance, richness, or relative dominance of resource species. Because bumble bees are highly mobile, resource variability in the surrounding landscape is also important for colonies and may mediate local-scale effects. We experimentally assessed the growth and reproduction of bumble bee colonies (Bombus impatiens) deployed in grasslands in different local- and landscape-scale resource environments. We found that floral dominance, rather than the overall abundance or richness of floral resources, was the most important local factor for colony growth and reproduction. This may reflect more efficient foraging on a few numerically dominant and abundant resource species. Local- and landscape-scale predictor variables had interacting effects on colony growth and reproduction, suggesting that foraging distance depends on where in the landscape efficiently used resources are located. Our results provide further evidence that conservation strategies aimed at enhancing floral resources can increase bumble bee population growth. However, the most effective form of floral enhancement may vary among bee species.

Cellulosic feedstock production on Conservation Reserve Program land: potential yields and environmental effects

Stephen D. LeDuc; Xuesong Zhang; Christopher M. Clark; César Izaurralde

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2017

Producing biofuel feedstocks on current agricultural land raises questions of a “food-vs-fuel” tradeoff. The use of current or former Conservation Reserve Program (CRP) land offers an alternative; yet the volumes of ethanol that could be produced and the potential environmental impacts of such a policy are unclear. Here, we applied the Environmental Policy Integrated Climate (EPIC) model to a U.S. Department of Agriculture database of over 200,000 CRP polygons in Iowa, USA, as a case study. We simulated yields and environmental impacts of growing three cellulosic biofuel feedstocks on CRP land: (i) an Alamo-variety switchgrass (Panicum virgatum L.); (ii) a generalized mixture of C4 and C3 grasses; (iii) and no-till corn (Zea mays L.) with residue removal. We simulated yields, soil erosion, and soil carbon (C) and nitrogen (N) stocks and fluxes. We found that although no-till corn with residue removal produced approximately 2.6-4.4 times more ethanol per area compared to switchgrass and the grass mixture, it also led to 3.9-4.5 times more erosion, 4.4-5.2 times more cumulative N loss, and a 10% reduction in total soil carbon as opposed to a 6-11% increase. Switchgrass resulted in the best environmental outcomes even when expressed on a per liter ethanol basis. Our results suggest planting no-till corn with residue removal should only be done on low slope soils to minimize environmental concerns. Overall, this analysis provides additional information to policy makers on the potential outcome and effects of producing biofuel feedstocks on current or former conservation lands. This article is protected by copyright. All rights reserved.

Co-evolution of domain interactions in the chloroplast TGD1, 2, 3 lipid transfer complex specific to Brassicaceae and Poaceae plants

Yang Yang; Agnieszka Zienkiewicz; Anastasiya Lavell; Christoph Benning

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2017

The import of lipids into the chloroplast is essential for photosynthetic membrane biogenesis. This process requires an ABC transporter in the inner envelope membrane with three subunits, TRIGALACTOSYLDIACYLGLYCEROL (TGD) 1, 2, and 3 named after the oligogalactolipids that accumulate in the respective Arabidopsis thaliana mutants. Unlike Arabidopsis, in the model grass Brachypodium distachyon, chloroplast lipid biosynthesis is largely dependent on imported precursors, resulting in a characteristic difference in chloroplast lipid acyl composition between plants. Accordingly, Arabidopsis is designated as a 16:3 (acyl carbons: double bounds) plant and Brachypodium as an 18:3 plant. Repression of TGD1 (BdTGD1) in Brachypodium affected growth without triggering oligogalactolipid biosynthesis. Moreover, expressing BdTGD1 in the Arabidopsis tgd1-1 mutant restored some phenotypes but did not reverse oligogalactolipid biosynthesis. A 27-amino acid loop (L45) is solely responsible for the incomplete functioning of BdTGD1 in Arabidopsis tgd1-1. Co-evolutionary analysis and co-immunoprecipitation assays showed that the TGD1 L45 loop interacts with the Mycobacterial Cell Entry domain of TGD2. To explain the observed differences in oligogalactolipid biosynthesis between the two species, we suggest that excess monogalactosyldiacylglycerol derived from chloroplast-derived precursors in Arabidopsis tgd1-1 is converted into oligogalactolipids, a process absent from Brachypodium with reduced TGD1 levels, which assembles monogalactosyldiacylglycerol exclusively from imported precursors.

Combining genome-scale experimental and computational methods to identify essential genes in Rhodobacter sphaeroides

Brian T. Burger; Saheed Imam; Mattew J. Scarborough; Daniel R. Noguera; Timothy J. Donohue

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2017

Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival.

Comprehensive characterization of non‑cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX‑pretreated corn stover

Christa Gunawan; Saisi Xue; Sivakumar Pattathil; Leonardo da Costa Sousa; Bruce E. Dale; Venkatesh Balan

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2017

Contentious relationships in phylogenomic studies can be driven by a handful of genes

Xing-Xing Shen; Chris T. Hittinger; Antonis Rokas

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2017

Phylogenomic studies have resolved countless branches of the tree of life, but remain strongly contradictory on certain, contentious relationships. Here, we use a maximum likelihood framework to quantify the distribution of phylogenetic signal among genes and sites for 17 contentious branches and 6 well-established control branches in plant, animal and fungal phylogenomic data matrices. We find that resolution in some of these 17 branches rests on a single gene or a few sites, and that removal of a single gene in concatenation analyses or a single site from every gene in coalescence-based analyses diminishes support and can alter the inferred topology. These results suggest that tiny subsets of very large data matrices drive the resolution of specific internodes, providing a dissection of the distribution of support and observed incongruence in phylogenomic analyses. We submit that quantifying the distribution of phylogenetic signal in phylogenomic data is essential for evaluating whether branches, especially contentious ones, are truly resolved. Finally, we offer one detailed example of such an evaluation for the controversy regarding the earliest-branching metazoan phylum, for which examination of the distributions of gene-wise and site-wise phylogenetic signal across eight data matrices consistently supports ctenophores as the sister group to all other metazoans.

Cover crop root contributions to soil carbon in a no-till corn bioenergy cropping system

Emily E. Austin; Kyle Wickings; Marshall D. McDaniel; Philip Robertson; Stuart Grandy

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2017

Deciphering the role of the phenylpropanoid metabolism in the tolerance of Capsicum annuum L. to Verticillium dahliae Kleb.

Marta Novo; Cristina Silvar; Fuencisla Merino; Teresa Martínez-Cortés; Fachuang Lu; John Ralph; Federico Pomar

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2017

Verticillium dahliae is an economically relevant soilborne pathogen that causes vascular wilt in several crops, including pepper (Capsicum annuum). Fungal infection is usually visualized as a vascular browning, likely due to the onset of phenylpropanoid metabolism, which also seems to play a crucial role in the tolerance of some pepper varieties. In the current work, the potential function of distinct phenylpropanoid derivatives (suberin, lignin and phenolic compounds) in the pepper tolerance response against V. dahliae, was investigated. Histochemical and biochemical analyses ruled out suberin as a key player in the pepper-fungus interaction. However, changes observed in lignin composition and higher deposition of bound phenolics in infected stems seemed to contribute to the reinforcement of cell walls and the impairment of V. dahliae colonization. Most importantly, this is the first time that the accumulation of the hydroxycinnamic acid amide N-feruloyltyramine was reported in pepper stems in response to a vascular fungus. Fungitoxic activity for that hydroxycinnamate-tyramine conjugate was demonstrated as well.

Defining the diverse cell populations contributing to lignification in Arabidopsis stems

Rebecca A. Smith; Mathias Schuetz; Steven D. Karlen; David Bird; Naohito Tokunaga; Yasushi Sato; Shawn D. Mansfield; John Ralph; Lacey Samuels

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2017

Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

Ewelina Mnich; Ruben Vanholme; Paula Oyarce; Sarah Liu; Fachuang Lu; Geert Goeminne; Bodil Jørgensen; Mohammed S. Motawie; Wout Boerjan; John Ralph; Peter Ulvskov; Birger L. Møller; Nanna Bjarnholt; Jesper Harholt

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2017

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the beta-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized beta-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

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