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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

Design of biofuel supply chains with variable regional depot and biorefinery locations

Rex T.L. Ng; Christos T. Maravelias

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We propose a multi-period mixed-integer linear programming (MILP) model for the design and operational planning of cellulosic biofuel supply chains. Specifically, the proposed MILP model accounts for biomass selection and allocation, technology selection and capacity planning at regional depots and biorefineries. Importantly, it considers the location of regional depots and biorefineries as continuous optimization decisions. We introduce approximation and reformulation methods for the calculation of the shipments and transportation distance in order to obtain a linear model. We illustrate the applicability of the proposed methods using two medium-scale examples with realistic data.

BdCESA7 , BdCESA8 , and BdPMT  utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

Deborah L. Petrik; Cynthia L. Cass; Dharshana Padmakshan; Cliff E. Foster; John P. Vogel; Steven D. Karlen; John Ralph; John C. Sedbrook

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2016

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the beta-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

FUSCA3  activates triacylglycerol accumulation in Arabidopsis seedlings and tobacco BY2 cells

Meng Zhang; Xia Cao; Qingli Jia; John Ohlrogge

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2016

Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3g26790, FUS3), are associated with embryo maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50 fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased 3-4 fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation. This article is protected by copyright. All rights reserved.

A novel pathway for triacylglycerol biosynthesis is responsible for the accumulation of massive quantities of glycerolipids in the surface wax of bayberry (Myrica pensylvanica ) fruit

Jeffrey P. Simpson; John B. Ohlrogge

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2016

Bayberry fruits synthesize an extremely thick and unusual layer of crystalline surface wax that accumulates to 30% of fruit dry weight, the highest reported surface lipid accumulation in plants. The composition is also striking, consisting of completely saturated triacylglycerol, diacylglycerol and monoacylglycerol with palmitate and myristate acyl chains. To gain insight into the unique properties of Bayberry wax synthesis we examined the chemical and morphological development of the wax layer, monitored wax biosynthesis through [14C]-radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 MAG as the first glycerolipid intermediate. The kinetics of [14C]-DAG and [14C]-TAG accumulation and the regiospecificity of their [14C]-acyl chains indicated distinct pools of acyl donors and that final TAG assembly occurs outside of cells. The most highly expressed genes were associated with production of cutin, whereas transcripts for conventional TAG synthesis were >50-fold less abundant. The biochemical and expression data together indicate that Bayberry surface glycerolipids are synthesized by a previously unknown pathway for TAG synthesis that is related to cutin biosynthesis. The combination of a unique surface wax and massive accumulation may aid understanding of how plants produce and secrete non-membrane glycerolipids, and also how to engineer alternative pathways for lipid production in non-seeds.

A superstructure-based framework for simultaneous process synthesis, heat integration, and utility plant design

Lingxun Kong; Murat Sen; Carlos A. Henao; James A. Dumesic; Christos T. Maravelias

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2016

We propose a superstructure optimization framework for process synthesis with simultaneous heat integration and utility plant design. Processing units in the chemical plant can be modeled using rigorous unit models or surrogate models generated from experimental results or off-line calculations. The utility plant subsystem includes multiple steam types with variable temperature and pressure. For the heat integration subsystem, we consider variable heat loads of process streams as well as variable intervals for the utilities. To enhance the solution of the resulting mixed-integer nonlinear programming models, we develop (1) new methods for the calculation of steam properties, (2) algorithms for variable bound calculation, and (3) systematic methods for the generation of redundant constraints. The applicability of our framework is illustrated through a biofuel case study which includes a novel non-enzymatic hydrolysis technology and new separation technologies, both of which are modeled based on experimental results.

Accuracy of genomic prediction in switchgrass (Panicum virgatum  L.) improved by accounting for linkage disequilibrium

Guillaume P. Ramstein; Joseph Evans; Shawn M. Kaeppler; Robert B. Mitchell; Kenneth P. Vogel; Robin Buell; Michael D. Casler

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2016

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

Actionable knowledge for ecological intensification of agriculture

Willemien Geertsema; Walter A.H. Rossing; Douglas A. Landis; Felix J.J.A. Bianchi; Paul C.J. van Rijn; Joop H.J. Schaminée; Teja Tscharntke; Wopke van der Werf

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2016

Ecological intensification of agriculture (EI) aims to conserve and promote biodiversity and the sustainable use of associated ecosystem services to support resource-efficient production. In many cases EI requires fundamental changes in farm and landscape management as well as the organizations and institutions that support agriculture. Ecologists can facilitate EI by engaging with stakeholders and, in the process, by generating “actionable knowledge” (that is, knowledge that specifically supports stakeholder decision making and consequent actions). Using three case studies as examples, we propose four principles whereby science can improve the delivery of actionable knowledge for EI: (1) biodiversity conservation helps to ensure the delivery of ecosystem services, (2) management of ecosystem services benefits from a landscape-scale approach, (3) ecosystem service trade-offs and synergies need to be articulated, and (4) EI is associated with complex social dynamics involving farmers, governments, researchers, and related institutions. These principles have the potential to enhance adoption of EI, but institutional and policy challenges remain.

An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula 

Chan Ma Ha; Luis Escamilla-Trevino; Juan Carlos Ser Yarce; Hoon Kim; John Ralph; Fang Chen; Richard A. Dixon

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2016

Biochemical and genetic analyses have identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana (Vanholme et al., 2013b), although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only week esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species. This article is protected by copyright. All rights reserved.

Applications of constraint-based models for biochemical production

Cameron Cotten; Jennifer L. Reed

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2016

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.

Biosynthesis of the plant cell wall matrix polysaccharide xyloglucan

Markus Pauly; Kenneth Keegstra

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2016

Xyloglucan (XyG) is a matrix polysaccharide that is present in the cell walls of all land plants. It consists of a beta-1,4-linked glucan backbone that is further substituted with xylosyl residues. These xylosyl residues can be further substituted with other glycosyl and nonglycosyl substituents that vary depending on the plant family and specific tissue. Advances in plant mutant isolation and characterization, functional genomics, and DNA sequencing have led to the identification of nearly all transferases and synthases necessary to synthesize XyG. Thus, in terms of the molecular mechanisms of plant cell wall polysaccharide biosynthesis, XyG is the most well understood. However, much remains to be learned about the molecular mechanisms of polysaccharide assembly and the regulation of these processes. Knowledge of the XyG biosynthetic machinery allows the XyG structure to be tailored in planta to ascertain the functions of this polysaccharide and its substituents in plant growth and interactions with the environment. Expected final online publication date for the Annual Review of Plant Biology Volume 67 is April 29, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Cell wall composition and biomass recalcitrance differences within a genotypically diverse set of Brachypodium distachyon  inbred lines

Cynthia L. Cass; Anastasiya A. Lavell; Nicholas Santoro; Cliff E. Foster; Steven D. Karlen; Rebecca A. Smith; John Ralph; David F. Garvin; John C. Sedbrook

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2016

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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2016

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.

Characterizing phenol–formaldehyde adhesive curec hemistry within the wood cell wall

Daniel J. Yelle; John Ralph

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2016

Adhesive bonding of wood using phenol–formaldehyde remains the industrial standard in wood product bond durability. Not only does this adhesive infiltrate the cell wall, it also is believed to form primary bonds with wood cell wall polymers, particularly guaiacyl lignin. However, the mechanism by which phenol–formaldehyde adhesive integrally interacts and bonds to lignin within the cell wall remains unclear. We used recently developed solubilization methodologies in conjunction with two-dimensional 1H–13C solution-state NMR spectroscopy of ball-milled pine earlywood and latewood bonded assemblies to characterize the chemical modification of wood cell wall polymers after phenol–formaldehyde curing at various cooking times. The results showed that the highly alkaline resin at 140 °C decreased the frequency of the principal arylglycerol-β-aryl ether interunit linkage by eighty percent in earlywood and by twenty percent in latewood. The presence of newly formed diarylmethanes between guaiacyl lignin units and phenolic methylols was confirmed via NMR spectra of the aliphatic methylene and aromatic regions. The phenol–formaldehyde cure chemistry showed that o–p methylene bridges dominated in both earlywood and latewood cell walls, but the propensity of p–p substitution is higher in the latewood cell wall. Our results provide evidence for a simultaneous wood polymer degradation and guaiacyl unit C5 bond formation that occurs during phenol–formaldehyde curing. This competition may be necessary for developing good bond durability between the adhesive and wood.

Climate-smart soils

K.eith Paustian; Johannes Lehmann; Stephen Ogle; David Reay; Philip Robertson; Pete Smith

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2016

Soils are integral to the function of all terrestrial ecosystems and to food and fibre production. An overlooked aspect of soils is their potential to mitigate greenhouse gas emissions. Although proven practices exist, the implementation of soil-based greenhouse gas mitigation activities are at an early stage and accurately quantifying emissions and reductions remains a substantial challenge. Emerging research and information technology developments provide the potential for a broader inclusion of soils in greenhouse gas policies. Here we highlight ‘state of the art’ soil greenhouse gas research, summarize mitigation practices and potentials, identify gaps in data and understanding and suggest ways to close such gaps through new research, technology and collaboration.

CO2 uptake is offset by CH4 and N2O emissions in a poplar short-rotation coppice

Terenzio Zenone; Donatella Zona; Ilya Gelfand; Bert Gielen; Marta Camino-Serrano; Reinhart Ceulemans

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2016

The need for renewable energy sources will lead to a considerable expansion in the planting of dedicated fast-growing biomass crops across Europe. These are commonly cultivated as short-rotation coppice (SRC), and currently poplar (Populus spp.) is the most widely planted. In this study, we report the greenhouse gas (GHG) fluxes of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) measured using eddy covariance technique in an SRC plantation for bioenergy production. Measurements were made during the period 2010–2013, that is, during the first two rotations of the SRC. The overall GHG balance of the 4 years of the study was an emission of 1.90 (±1.37) Mg CO2eq ha−1; this indicated that soil trace gas emissions offset the CO2 uptake by the plantation. CH4 and N2O contributed almost equally to offset the CO2 uptake of −5.28 (±0.67) Mg CO2eq ha−1 with an overall emission of 3.56 (±0.35) Mg CO2eq ha−1 of N2O and of 3.53 (±0.85) Mg CO2eq ha−1 of CH4. N2O emissions mostly occurred during one single peak a few months after the site was converted to SRC; this peak comprised 44% of the total N2O loss during the two rotations. Accurately capturing emission events proved to be critical for deriving correct estimates of the GHG balance. The nitrogen (N) content of the soil and the water table depth were the two drivers that best explained the variability in N2O and CH4, respectively. This study underlines the importance of the ‘non-CO2 GHGs’ on the overall balance. Further long-term investigations of soil trace gas emissions should monitor the N content and the mineralization rate of the soil, as well as the microbial community, as drivers of the trace gas emissions.

Comparative productivity of alternative cellulosic bioenergy cropping systems in the North Central USA

Gregg R. Sanford; Lawrence G. Oates; Poonam Jasrotia; Kurt D. Thelen; Philip Robertson; Randall D. Jackson

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2016

Biofuels from lignocellulosic feedstocks have the potential to improve a wide range of ecosystem services while simultaneously reducing dependence on fossil fuels. Here, we report on the six-year production potential (above ground net primary production, ANPP), post-frost harvested biomass (yield), and gross harvest efficiency (GHE = yield/ANPP) of seven model bioenergy cropping systems in both southcentral Wisconsin (ARL) and southwest Michigan (KBS). The cropping systems studied were continuous corn (Zea mays L.), switchgrass (Panicum virgatum L.), giant miscanthus (Miscanthus × giganteus Greef & Deuter ex Hodkinson & Renvoize), hybrid poplar (Populus nigra × P. maximowiczii A. Henry ‘NM6’), a native grass mixture (5 sown species), an early successional community, and a restored prairie (18 sown species). Overall the most productive cropping systems were corn > giant miscanthus > and switchgrass, which were significantly more productive than native grasses ≈ restored prairie ≈ early successional ≈ and hybrid poplar, although some systems (e.g. hybrid poplar) differed significantly by location. Highest total ANPP was observed in giant miscanthus (35.2 ± 2.0 Mg ha−1 yr−1) at KBS during the sixth growing season. Six-year cumulative biomass yield from hybrid poplar at KBS (55.4 ± 1.3 Mg ha−1) was high but significantly lower than corn and giant miscanthus (65.5 ± 1.5, 65.2 ± 5.5 Mg ha−1, respectively). Hypothesized yield advantages of diversity in perennial cropping systems were not observed during this period. Harvested biomass yields were 60, 56, and 44% of ANPP for corn, perennial grass, and restored prairie, respectively, suggesting that relatively simple changes in agronomic management (e.g. harvest timing and harvest equipment modification) may provide significant gains in bioenergy crop yields. Species composition was an important determinant of GHE in more diverse systems. Results show that well-established, dedicated bioenergy crops are capable of producing as much biomass as corn stover, but with fewer inputs.

Complex ancestries of lager-brewing hybrids were shaped by standing variation in the wild yeast Saccharomyces eubayanus 

David Peris; Quinn Langdon; Ryan V. Moriarty; Kayla Sylvester; Martin Bontrager; Guillaume Charron; Jean-Baptiste LeDuc; Christian R. Landry; Diego Libkind; Chris T. Hittinger

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2016

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.

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