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December 31, 2015

Comparison of cell wall (1) and growth (2) traits between T2-generation transgenic (+) and non-transgenic null-segregant (-) hybrid families.

Successful Transfer of Transgenic Switchgrass Low-Recalcitrance Traits into High-Yielding Field Variety
This study evaluated the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits and to preserve the transgenic benefit. Our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.

December 31, 2015

GPC Curves

BESC Biomass Improvements In Lignin Extended From Grasses To Eucalyptus
Lignin reduction through breeding and genetic modification has the potential to reduce costs in biomass processing. Previous work demonstrated that both C3′H and C4H down-regulated lines had lower S/G ratios, lignin and recalcitrance. Both lines have significantly higher extractability under NaOH base extraction, indicating altered cell wall construction. Although the C3′H contained more H lignin than the C4H and control samples, only slight reductions in average molecular weights of the lignin and few changes in the lignin subunit were observed.

December 29, 2015

Preferential expression of FPGS1 in the vascular tissues of Arabidopsis.

Reducing Recalcitrance In Plants By Down-Regulating A Gene Involved In C1 Metabolism
Folylpolyglutamate synthetase (FPGS) genes and their corresponding mutants were characterized in Arabidopsis using cellular, biochemical, genomic and molecular approaches. Down-regulation of a single FPGS isoform leads to reduced lignin and altered hemicellulose in plants and therefore should be explored further for the improvement of lignocellulosic feedstocks.

December 26, 2015

Root enhancement in winter wheat due to S. vermifera colonization in vitro.

Review: Potential Impact of S. vermifera, A Unique Root Symbiont, On Agronomics of Biofeedstocks
Research performed with orchid mycorrhizal fungus Sebacina vermifera clearly indicates significant growth-promoting abilities in a remarkably broad spectrum of plants, including switchgrass, that rivals or even surpasses that of arbuscular mycorrhizae. Sebacinoid fungi should be considered as a previously hidden, but amenable and effective microbial tool for enhancing plant productivity and stress tolerance which has been validated in switchgrass, an important biofeedstock for biofuels.

December 14, 2015

System-wide Redox Stress Response: red indicates decreased transcription, (#) is the number of genes in the pathway decreased.

Towards Understanding Gene Systems Mediating Redox Homeostasis and Energy Metabolism in C. thermocellum
Current moderate ethanol yields and productivities as well as growth inhibition impede industrial deployment of this bacterium for commodity fuel production. Redox imbalances may contribute to incomplete substrate utilization, limit bioproductivity, and direct fermentation products to undesirable overflow metabolites. The redox systems identified are active and fungible in C. thermocellum upon introduction of a redox stress, and considered a link between redox stress and the occurrence of ethanol yield-limiting, unwanted metabolic processes. This expression study informs ongoing metabolic engineering efforts to increase bioproductivity in C. thermocellum.

December 10, 2015

Commercial Licensing of a BESC Global Genetic Regulator of Aromatic Biosynthesis
BESC has been studying SNPs and key compositional and conversion phenotypes in the natural variation population in Populus using GWAS (genome-wide association studies). A study of rare SNPs identified an unusual paralog in Populus which had taken on new regulator functions. Patent applications have been filed. Follow-on work is testing the utility in other plant species. The technology is being licensed to two companies who plan to commercialize the technology: GreenWood Resources and Forest Genetics International. Increasing biofuel yield or forage digestibility can lead to reductions in land use for these crops and thus contributing to long term sustainability.

October 26, 2015

Integrating mRNA and Protein Sequence Data to Enable Detection and Quantitative Profiling of Natural Protein Sequence Variants in Populus
In this study, we used next-generation RNA-sequencing data to generate genotype-specific protein databases, which allowed the identification and quantification of sequence variations at the protein level. A variety of natural sequence protein variants were quantified in two Populus trichocarpa genotypes. In addition to expected neutral single amino acid polymorphisms, investigators characterized polymorphisms predicted to be non-neutral and located in regions of the genome predicted to have undergone recent positive and/or divergent selection and therefore represent a candidate list of protein variants relevant to plant adaptability.

October 16, 2015

Direct Catalytic Upgrading of Ethanol is Efficient and Works Through a Hydrocarbon Pool Mechanism
Experiments with C2H5OD and in situ DRIFTS (diffuse reflectance FT infrared spectroscopy) suggest that most of the products come from the hydrocarbon pool type mechanism and endothermic dehydration step is not necessary. This mechanism supports the slightly exothermic nature of the reactions and potentially improves the process economics.

October 13, 2015

Metabolic Model Used to Predict and Remove Redox Bottlenecks in C. thermocellum Biofuel Production
Metabolic engineering strategies have been used to enhance ethanol production in C. thermocellum, a highly effective cellulose degrader. A central metabolic model has been constructed and validated for C. thermocellum which can expedite the process of designing and testing metabolic engineering strategies. Using the model for experimental design, investigators were able to highlight key overexpression targets for metabolic engineering such as RNF, NFN, or AdhE and to predict genotypes which may enhance ethanol yield.

October 13, 2015

Effect of glucan and cellulase loadings on ethanol yield for CELF-prepared corn stover

CELF Pretreatment Boosts Biofuel Yields and Titers
Novel Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment has shown to be highly effective in achieving near theoretical sugars yields for corn stover. Earlier preliminary data also showed that CELF performs much better than dilute acid (DA) pretreatment; however, the effect of solids concentrations and enzyme loadings on ethanol yields and titers was not known. The results reported show that CELF pretreatment makes possible high ethanol yields and titers, and most importantly, at low enzyme loadings.

October 12, 2015

Molecular Dynamics Simulation Reveals Molecular Basis of Highly Efficient Lignin Solubilization
Previous BESC work has shown that pretreatment of biomass with a tetrahydrofuran (THF)–water cosolvent is highly effective at extracting lignin from biomass and achieving high yields of fermentable sugars from cellulose. However, the molecular mechanism of this effect is not known. In most aqueous-based pretreatments, lignin is not removed entirely from biomass because it aggregates onto the cellulose surface, blocking enzymatic access to cellulose. Under theta solvent conditions, polymers do not aggregate, thus providing a mechanism for the observed lignin solubilization that facilitates unfettered access of cellulolytic enzymes to cellulose. This mechanism may provide insights to improve this or other novel pretreatments.

September 22, 2015

Computational Inference of the Structure and Regulation of the Lignin Pathway in Switchgrass, Panicum virgatum
Recalcitrance is partially due to the heterologous polymer lignin. The biosynthetic pathway leading to monolignols, and thus to lignin, is not completely known in switchgrass, and difficulties associated with in vivo measurements of its intermediates pose a challenge for a true understanding of the functioning of the pathway. However, such an understanding is a prerequisite for manipulations of transgenics toward altered lignin properties and a reduction in recalcitrance. The computational analysis suggests regulatory mechanisms of product inhibition and enzyme competition, which are well known in biochemistry, but so far had not been reported in switchgrass. Our deepened understanding of the pathway allows us to make prediction regarding combined knock-downs that are likely to alter lignin properties significantly.

September 01, 2015

Highest Yield to Date of Ethanol from Engineered Clostridium thermocellum
C. thermocellum is one of the fastest growing anaerobic organisms on crystalline cellulose. Metabolic engineering is required to increase ethanol yield by eliminating pathways to other fermentation products. Effectively eliminated lactate, acetate, formate, and most H2 production. Ethanol yield doubled relative to the wild type, both on model substrates and dilute-acid pretreated plant biomass. Allowed for higher titer ethanol production from cellulose in the absence of pH control. Highest ethanol yield achieved to date in C. thermocellum. Created platform strain for further metabolic engineering for consolidated bioprocessing.

September 01, 2015

Engineering of a Thermophilic, Cellulolytic Microbe to Produce an Advanced Biofuel
Production of isobutanol from cellulose in minimal medium achieved 5.4 g/L titer and roughly 41% of theoretical yield. This result successfully demonstrates the advantages of using a consolidates bioprocessing approach to produce an advanced biofuel. This is a measureable and significant step towards commercially relevant titers and yields. Isobutanol can be utilized as a "drop-in" biofuel, as a substrate for upgrading into hydrocarbon fuels, or as a feedstock for other bioproducts.

August 29, 2015

Mechanism for expression of xylanases induced by extracellular xylan via sig16

New Insights into Regulation of Cellulosomal Xylanase Genes in Clostridium thermocellum
Clostridium thermocellum is regarded as a model cellulolytic thermophile, and can grow well on un-pretreated lignocellulosic feedstock, yet it does not utilize C5 sugars (e.g., xylose, arabinose) despite including xylanases in its cellulosome. While unable to utilize C5 sugars C. thermocellum has a set of sigma factor regulated xylanases that, when expressed in response to biomass, can increase the amount of solubilized carbohydrate including glucan.

August 29, 2015

BESC co-inventors Sara Jawdy and Lee Gunter evaluating growth performance of rice plants carrying the lignin reducing-flavonoid enhancing mechanism.

Forage Genetics Plans to Commercialize BESC Invention
The invention provides a genetic mechanism for the reduction of lignin biosynthesis while increasing concentration of desirable flavonoids. Reduced lignin content increases digestibility and nutritional value of animal feedstocks such as alfalfa, corn and sorghum. Forage Genetics plans to evaluate commercial viability of this technology in alfalfa, corn and sorghum forage crops as animal feedstocks.

August 27, 2015

Sugar Release with 17.5mins Pretreatment at 180C

Application of Results from Down-Regulation of Lignin in Populus and Switchgrass Leads to Similar Effects in Eucalyptus
Changes to gene expression in switchgrass and Populus have shown increased sugar release and reduced recalcitrance. This work sought to transfer results to Eucalyptus, a fast-growing warm climate woody biofeedstock also suitable for cellulosic biofuel production. This work extends result that lowering overall lignin content, rather than altering S/G/H ratios, has the largest impact on reducing recalcitrance in eucalyptus.

August 01, 2015

Expression and Secretion of E1 in C. bescii Enhances the Cellulolytic Ability of Its Secretome
The in vitro activity of the C. bescii secretome was significantly increased (17.5% at 65oC) by the addition of E1. The E1 protein acts synergistically with CelA to digest cellulose and this result offers the possibility of adding enzymes into engineered secretomes for improved biomass deconstruction. This work contributes to an understanding of the action and limitations of the CelA enzyme that will enable enzyme engineering to generate novel enzymes mixtures for biomass deconstruction.

July 27, 2015

Discovery of Novel Tungsten-Utilization Pathway in Caldicellulosiruptor bescii
Very few microbes are known to require tungsten for growth. Analysis of the genome sequence of C. bescii has unexpectedly revealed a set of transporter genes that are highly specific for the uptake of tungsten. Although its role within the cell is as yet unknown, the conservation of XOR and associated pyranopterin synthesis genes across the genus Caldicellulosiruptor, suggest that this novel tungstoenzyme serves an important role in the primary metabolism of these cellulolytic species.

July 20, 2015

Catalytic Mechanism and Unique Low pH Optimum of Caldicellulosiruptor bescii Pectate Lyase Explained via Crystallography and Mutagenesis
Pectate lyases are a degradation enzyme for an important if small polymer component of plant biomass. The structural changes upon all mutations are analysed and their effect on activity explained. This paper provide a comprehensive examination on the PL family 3 structure and function with an extensive mutagenesis study.

July 15, 2015

Comparison of MethylC-seq and 4mC-TAB-seq.

Rapid Identification of Restriction Systems that Prevent DNA Transformation of Uncharacterized Bacteria
New 4mC-specific sequencing strategy will help uncover and facilitate investigation of 4mC in bacterial R-M systems and can advance the rapid and efficient genetic engineering of these bacteria in the future.

July 01, 2015

Imaging with HPFM (a,c,e,g) complemented with confocal Raman (b,d,f,h) cross-section of fresh (a,b), (...more)

New Hybrid Microscope Provide Unparalleled Submicron View of Plant Cell Walls
A novel, noninvasive method to explore submicron structures of biomass before and after treatment. Researchers will be able to study samples ranging from naturally occurring biological polymers, tissues and plant cells to engineered nanoparticles and nanostructures.

June 22, 2015

Green: Fungal mycelia Red: Root cell membrane

Novel Delivery System for Root Symbiotic Fungus, Sebacina vermifera, Enhances Biomass yield of Low Lignin Switchgrass Lines
S. vermifera colonization enhanced plant biomass growth by 15-50% independent of delivery method; however, clay-based delivery system increased the percentage of fungal biomass in planta. Root biomass and fungal colonization was significantly improved with clay-based system.

June 08, 2015

Transport energy use by fuel and year, displaced CO2 emissions by fuel and year, and tota (...more)

Making the Case for Why Biofuels are Needed to Achieve Low-Carbon Transport Through 2050 and Beyond
Even with aggressive reductions in travel growth, shifts to mass transport modes, strong efficiency improvements and deep market penetration by electricity and hydrogen, there remains a large demand for dense liquid fuels: 80% of transport energy demand in 2050, and 50% in 2075. If conventional fossil fuels were used to meet this demand, emissions from the transportation sector would exceed the economy-wide emissions consistent with the two degree scenario. It will likely be difficult to achieve a low-carbon transport sector without widespread use of biofuels.

June 01, 2015

Kevin Wenger of Mascoma, a subsidiary of Lallemand, LLC and Paul Gilna of BESC

BESC Partner Mascoma Launches C5 FUEL™ — A Yeast Engineered for Improved Cellulosic Biofuel Production
The new strain of yeast simultaneously yields 97 percent conversion of xylose – the C5 sugar – and glucose and it accomplishes this feat in less than 48 hours. The launch of a commercially available product designed for producers in the cellulosic biofuels market is an important milestone for BESC and the BRC program.

May 29, 2015

Book edited by BESC Scientist on Consolidated Bioprocessing
Mike Himmel has edited a new book on "Direct Microbial Conversion of Biomass to Advanced Biofuels," Elsevier, London/New York, NY. 2015. Several chapters report on BESC work and acknowledge BER support.

May 14, 2015

Perspective: Potential Impact of Modern Biotechnology on Future Supply of Biofuels
This study assesses the feasibility and advantages of near-future (10-year) biotechnological developments for a U.S. biomass-based supply chain for bioenergy production.

April 27, 2015

DOE Bioenergy Research Centers Well Represented at the 37th Symposium on Biotechnology for Fuels and Chemicals — 2015
19% of oral presentations were from BESC, GLBRC and JBEI (26 out of 138) and approximately 27 BRC posters were presented. There were 650 attendees from academia, industry and government.

April 15, 2015

Glucose Regression Model

Pyrolysis Molecular Beam Mass Spectrometry as a High-throughput Method for Determining Biomass Sugar Content
Py-MBMS can be used to estimate sugar content of biomass using regression models. The method is much faster (< 2 min/sample) than traditional hydrolysis followed by HPLC with much less sample preparation. This method introduces a new, high-throughput technique for sugar compositional analysis. The py-MBMS spectra can also be used to estimate lignin and S/G ratio, providing a comprehensive analysis.

April 01, 2015

Dr. Liao received his Bachelor of Science degree at the National Taiwan University and his Doctorate at the University of Wisconsin-Madison

James Liao Elected to the National Academy of Sciences
Dr. Liao's science focuses on developing the biological production of the liquid fuel isobutanol while simultaneously consuming carbon dioxide and developing different methods to create liquid fuels from electricity and from waste proteins; as well as identifying a more efficient way to convert sugars into fuels. Dr. Liao serves as the BESC Activity Lead for Butanol Production by CBP.

March 31, 2015

Wild type cellulase, CbhA; Chimeric cellulase with swapped linker domain.

Improved multi-functional cellulase awarded US Patent
One of the most abundant cellulase components of the CipA scaffold, CbhA, was improved by replacing the Fn3 domains with a thermal stable linker peptide from another cellulolytic microbe. Changing out the linker domain doubled the activity of the CbhA enzyme. U.S. Patent 8993276 (March 31, 2015) was issued for the invention of the new improved cellulase enzyme.

March 21, 2015

New Promoters for Metabolic Engineering of C. thermocellum
Several promoters were identified (eno, cbp, cbp_2, 815, 966, 2638 and 2926) which gave high expression and high enzymatic activity of both reporter genes in C. thermocellum.

March 16, 2015

Release of a genome dataset comprised of results from multiple sequencing modalities
Access to a comprehensive dataset representing a range of sequencing technologies can serve as a benchmark/training dataset to assist others in developing new technologies and computational algorithms.

March 05, 2015

Review: Ethanol Production by Engineered Thermophiles
Four potential pathways from the glycolysis intermediate pyruvate into ethanol have been identified: pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), pyruvate ferredoxin oxidoreductase (PFOR) and pyruvate formate lyase (PFL). Three of these pathways (PDC, PDH and PFOR) have been used in thermophiles for ethanol production at significantly higher yield, and two pathways (PDC and PFOR) resulted in ethanol production at higher titer.

March 01, 2015

Reducing Recalcitrance and Increasing Growth in Populus by Downregulation of Matrix Polysaccharide Biosynthetic Gene
Populus GAUT12.1-Knockdown lines have 3 to 7% increased sugar release, confirming strategy to reduce recalcitrance. Populus GAUT12.1-Knockdown lines have 12 to 52% increase in plant height, 12 to 44% increased stem diameter, and 52 to 117% increased leaf size, demonstrating that plant growth and recalcitrance are linked and can simultaneously be improved. Improved biomass phenotype was accompanied by reduction in pectin and xylan cell wall content, supporting hypothesis that pectin and xylan contribute to a wall infrastructure(s) critical for recalcitrance.

March 01, 2015

Tāpirins, a New Class of Biomolecules, Mediate Attachment of Caldicellulosiruptor Species to Cellulose
Tāpirins represent a new class of proteins used by Caldicellulosiruptor species to attach to insoluble cellulose. The tapirins establish a new paradigm for how cellulolytic bacteria adhere to cellulose.

February 27, 2015

Genomes Sequenced

Review paper summarizes insights from 20 years of bacterial genome sequencing
Second and third generation sequencing have greatly reduced the cost of sequencing and made it accessible to a great many more individual investigators. Sequencing of bacterial genome sequences is now a standard procedure and the information from tens of thousands of bacterial genomes has had a major impact on the view of the bacterial world.

February 21, 2015

Ten initial BESC Populus TOP lines identified with attractive biofuel traits in conversion and growth
Simultaneous comparison of Populus TOP lines for enhanced growth and biofuel traits will allow BESC researchers to identify the best among them to further evaluate in field studies (e.g., to determine sustainability traits) in preparation for application and licensing with commercial partners.

February 11, 2015

Novel Pretreatment Greatly Reduces Enzyme Loadings and Increases Biofuel Yields
Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment achieved about a 10-fold reduction in enzyme loadings compared to traditional DA pretreatment. CELF also removed a majority of biomass lignin (over 90% in wood) that could be precipitated as a high purity product suitable for conversion to high value chemicals and materials.

February 01, 2015

Glycome Profiling of AFEX™ Pretreated Biomass Provides Insights into Plant Cell Wall Structure
AFEX™ pretreatment, in general, causes loosening of non-cellulosic glycans, especially xylans, in diverse biomass types resulting in a reduced recalcitrance. These effects of AFEX™ vary significantly depending on the type of biomass. Monitoring changes in cell wall glycan compositions and their relative extractability for untreated and pretreated plant biomass provide improved understanding of variations in structure and composition of plant cell walls that relate to the role(s) of matrix-polysaccharides in recalcitrance.

January 30, 2015

Production of lignofuels and electrofuels by extremely thermophilic microbes
Caldicellulosiruptor and Pyrococcus sp. could be potentially metabolically engineered to produce advanced biofuels compatible with existing infrastructure, generating so-called lignofuels and electrofuels, respectively. Thermophilic biomass degradation is more efficient, and thermophilic fuel synthesis affords the opportunity for simultaneous distillation of volatile products.

January 30, 2015

Novel approach to blocking H2 production increases Clostridium thermocellum ethanol yield
Approach allowed four hydrogenases to be simultaneously inactivated using only two mutations on the chromosome. Highest C. thermocellum ethanol yield reported so far shows promise for further yield improvement. Provides insight into redox balancing in C. thermocellum. Mutation demonstrates ethanol tolerance in C. thermocellum is more complicated than previously thought.

January 25, 2015

Field experiment in the second (2013) growing season. Photographed August 1 2013

Field performance of modified switchgrass demonstrates a 'Goldilocks Effect'
This study showed that too high PvMYB4 transgene expression is fatal to plants in the field, whereas too low expression shows no difference from controls; but when optimal expression is achieved the altered switchgrass produces higher biomass and biofuel yields. These gains represent the potential for a doubling of biofuel production per hectare over conventional feedstocks. This is the highest potential gain reported to date from any field-grown modified feedstock.

January 23, 2015

A four-year-old P. trichocarpa in a common garden

Rapid gene discovery and validation using the BESC Populus association population
Complementary QTL and association mapping are powerful tools for rapid gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

January 11, 2015

BESC Novel Approach to Bioenergy Outreach and Education
BESC and the Creative Discovery Museum have developed and implemented a novel 'hub and spoke' model by working with science museums and centers around the country to deliver impactful science outreach in biofuels. The program continues to move toward becoming self-sustaining with over 75% of the hands-on interactions being supported by local schools and museums.

January 01, 2015

Pore size distribution of Populus before and after dilute acid pretreatment measured by Mercury porosimetry.

The Role of Nano-Porosity in Lowering Recalcitrance
Pore size distribution analysis indicated that the most fundamental barrier in terms of the biomass porosity scale for efficient enzymatic hydrolysis is the nano-pore space formed between coated microfibrils, although some of the porous architecture such as the cell lumen and pit could be severely destroyed after pretreatment. Lignin still plays a negative role in the processes of enzymatic hydrolysis by binding to cellulases; but this negative effect could be compensated by the positive effect of cellulose accessibility.

January 01, 2015

BioEnergy Research Vol. 8(3), 2015.

BioEnergy Research Special Issue Features DOE Bioenergy Research Centers
Sixteen articles are being released in a special issue devoted entirely to the work of the three DOE Bioenergy Research Centers including an overview of the three Centers and articles on feedstock sustainability, pretreatment and fermentation.

BioEnergy Science Center one of three DOE Bioenergy Research Centers established by the U.S. Department of Energy.