Highlights Archive

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December 22, 2016

RNA-Seq analysis for man5A (mannanase) region in ΔglyR2 mutant and parent strains

Identification of LacI Repressor-Based Regulatory Network Activity Targeting Hemicellulases in C. thermocellum
Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. We combined use of three lacI gene deletions with transcriptomics and DNA binding assays to gain insights into LacI regulatory networks. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

December 22, 2016

Correlation of sugar release with lignin content

Key Traits Leading to Reduced Recalcitrance Remain Stable Following Three Years of Field Trials
Down-regulation of the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass has been previously shown by BESC to improve the thermochemical and biochemical conversion of biomass. The originally reported improvements in biomass cellulose accessibility wrought by down-regulation of COMT in switchgrass have remained stable following three years of field trials. Demonstrating stability of introduced traits for improved biofuel production in bioenergy crops is an important proof-of-principle on the path toward long-term validation of such approaches.

December 12, 2016

A representative HSQC NMR spectra of natural poplar variant lignin

Use Of Populus Natural Variants As A Research Tool To Further Determine Molecular Basis Of Recalcitrance
This project investigates the relative contributions of various factors to recalcitrance by a first-order multi-variants linear correlation analysis, which significantly helps improve the understanding of the fundamental mechanisms of biomass recalcitrance.

December 06, 2016

Metabolic network

Confirmation of Role for Malate Shunt in Clostridium thermocellum Glycolysis
The unique metabolism of C. thermocellum has presented difficulties in engineering it for improved ethanol production. Improved understanding of the malate shunt will allow the design of strains for improved ethanol production.

November 29, 2016

In situ 3D SRS imaging to track xylan distribution in deacetylated corn stover cell wall before and after xylan digestion

New Approach Allows Resolution Of Xylan Polymers In Plant Cell Wall
This approach demonstrated that xylan, an important plant polymer source, can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy. This can be used to enhance understanding of xylan in cell wall biosynthesis and deconstruction.

November 25, 2016

Comparison of biomass solubilization by C. thermocellum relative to fungal cellulases. The da (...more)

State of Play in Ethanol Production using Consolidated Bioprocessing with C. thermocellum and T. saccharolyticum
A comprehensive book chapter that addresses Consolidated Bioprocessing (CBP) organism development strategies; plant cell wall solubilization by C. thermocellum; bioenergetics of C. thermocellum cellulose fermentation; and metabolic engineering and the current state of strain development.

November 21, 2016

Simplified structure of plant cell walls (A), lignin isolated from poplar (B), and schematic structure of poplar lignin (C)

Mini Review Surveys Improvements In Understanding Relationship Of Lignin Structure To Biomass Recalcitrance
This review focuses on advances in understanding the specific roles of lignin properties during pre-treatment; lignin-enzyme interactions; monolignol compositional units; and hydroxycinnamates and hydroxyl and carboxylic groups in lignin.

November 03, 2016

Comparisons of five transgenic switchgrass lines to their parental controls (* indicates not a stati (...more)

Comparative Analysis Of Field-Grown Transgenic Switchgrass Lines Shows Stable Increases In Biofuel Yields
Clones of plants representing independent transgenic events and their respective non-transgenic control lines were investigated for biomass yield, carbohydrate composition, and recalcitrance to bioconversion via separate hydrolysis and fermentation to ethanol. Over two consecutive field-growth seasons, most transgenic lines maintain higher glucan and xylan yield at similar, or sometimes better, plant biomass. Transgenic lines targeting cell wall modifications yielded significantly higher bioconversion to ethanol (up to 36% and 21% in year 1 and year 2, respectively).

November 02, 2016

Mean soil organic matter in the upper 0-15 cm (A) and deeper 15-30 cm (B) during the first two growi (...more)

Field Grown Transgenic Switchgrass Has No Negative Affect On Soil Chemistry, Microbiology Or Carbon Storage Potential
BESC has demonstrated that when shoot S/G lignin ratio is decreased in switchgrass, greater yields of biofuel can be produced; understanding agronomic consequences of such changes is important to proving industrial value of engineered biofuel crops. COMT-altered switchgrass appears to be substantially equivalent to non-engineered switchgrass with regards to soil and microbiome properties.

November 01, 2016

Heat map of C. thermocellum transcriptomic response to exogenous acetate. Blue and yellow ind (...more)

Improving Robustness of Engineered C. thermocellum
Understanding and overcoming low robustness in engineered microorganisms will be essential to industrial deployment of these organisms. This study helps reveal mechanisms of electron balancing in C. thermocellum that leads to increased robustness in its potential use in biofuel production.

November 01, 2016

Diagram of electron metabolism in C. thermocellum

Engineering Electron Metabolism to Increase Biofuel Production in Clostridium thermocellum
Engineering electron metabolism is a promising strategy for improving ethanol production in C. thermocellum. A strain with improved ethanol production was also generated by overexpressing the rnfCDGEAB operon.

October 28, 2016

Schematic of reductive C1 metabolism initiated from reversed PFOR and PFL. Carbons in red indicate < (...more)

C. thermocellum Endowed with Ability to Fix CO2
A novel route that C. thermocellum employs to fix CO2 when grown in primarily heterotrophic mode supplemented with sodium bicarbonate was discovered. Critical enzymes responsible for fixing CO2 and channeling the fixed carbon to the C1 metabolic pathway were identified. This research paves the way to future engineering of the Clostridium thermocellum bacterium to utilize cellulose and CO2 simultaneously as a means to improve microbial carbon efficiency and reduce CO2 in the environment.

October 19, 2016

National Bioenergy Day
BESC participated with seven different agencies to provide educational and informative biofuel connected activities and displays for ~200 elementary children during the National Bioenergy Day celebration held at the University of Tennessee (UT) Arboretum in Oak Ridge, Tennessee.

September 30, 2016

GreenWood and ORNL Sign Agreement.
Seated: Brian Stanton, Chief Science Officer, GreenWood Resour (...more)

GreenWood Resources Licenses BESC Invention to Boost Biofuel Yield
GreenWood Resources has licensed a BESC technology based on the discovery of a gene in Populus trichocarpa that makes it easier to convert poplar trees into biofuels. They plan to commercialize the technology to select and breed better varieties of poplar with less lignin content, which simplifies the conversion process and ultimately lowers the overall costs of biofuel production.

September 15, 2016

New Thioacidolysis Method Allows for Improved Throughput in Linking Biomass Recalcitrance with Lignin Structure and Composition
Thioacidolysis is a method used to measure the relative content of coumaryl (H), syringyl (S) and coniferyl (G) alcohol lignin monomers bound by β-O-4 linkages. This new method was developed with significantly higher throughput than traditional techniques and was found to measure statistically similar S/G ratios in lignin from different lignocellulosic biomass types relative to a traditional low-throughput technique. The composition and structure of lignin based on the relative amount of monomers bound by β-O-4 linkages can be more rapidly determined for screening purposes allowing for improved throughput in linking biomass recalcitrance with lignin structure and composition.

September 06, 2016

GEM allows for reaction level investigation

Development Of A Genome-Scale Metabolic Model Of C. Thermocellum Assists In Strain Design For Biofuels And Chemicals
Genome scale modeling is a powerful tool for investigating cellular metabolism. A genome scale model (GEM) of DSM 1313 was constructed and curated for simulation of growth on alternative cellulosic substrates by implementing an adjustable cellulosome. Using the GEM, researchers were able to explore how key redox and bioenergetic reactions change as a function of growth rates and carbon sources in a systematic manner. Use of the model led to a proposed substrate-dependent regulatory mechanism by which ethanol production is enhanced or limited.

August 25, 2016

Heterologous Expression of Xylanases from A. cellulolyticus Enhances Hemicellulolytic Activity of C. bescii
To test whether the addition of A. cellulolyticus xylanases might enhance the hemicellulolytic activity of C. bescii and growth on xylan substrates, the xylanases were expressed in C. bescii using new expression vectors. Expression of the xylanases in C. bescii results in an increase in the activity of the exoproteome on xylan and a dramatic increase in its ability to grow on xylan substrates.

August 10, 2016

Microbial Upgrading of Lignin-Derived Carboxylates to Branched-Chain Esters for Drop-in Biofuels
An innovative biological conversion route was developed for conversion of lignocellulosic biomass or organic wastes into biofuels and high-value chemicals.

August 08, 2016

Cellulose in THF-Water

HPC Simulation of Co-Solvent Pretreatment of Biomass Confirmed by Experiment
Previous BESC work has shown that reaction of biomass in aqueous tetrahydrofuran (THF) in CELF (Co-solvent Enhanced Lignocellulosic Fractionation) pretreatment is highly effective at delignification and deconstruction of biomass for biofuels and bioproducts. Molecular dynamics simulations were performed of whole cellulose fibers and single cellulose chains in a THF-water co-solvent at various temperatures on the ORNL TITAN supercomputer. The simulations were complemented by THF-water experiments on cellulose, measuring its solubilization and imaging the product residue.

August 05, 2016

Creative Discovery Museum Receives Pinnacle Award for Distance Learning
The Creative Discovery Museum (CDM) has been awarded a Center for Interactive Learning and Collaboration (CILC) Pinnacle Award for 2015 – 2016 based on recipient evaluations. The BESC-sponsored CDM "Distance Learning Biofuels" program is a 50-minute lesson designed for students in grades 4 – 8. A museum educator from CDM provides the lesson via video conferencing. The program provides the opportunity for students to learn about innovative research taking place in the fields of biofuels and alternative energy.

July 27, 2016

One-hour hydrolysis rate versus cumulative cellulose conversion for uninterrupted and restarted hydr (...more)

Cellulose DP And Accessibility Contribute To Cellulase Enzyme Action On Biomass Substrate
The enzymatic hydrolysis of lignocellulosic biomass proceeds initially at a fast rate followed by a rapid decrease in the conversion rates. Degree of polymerization (DP) analysis during hydrolysis suggested a synergistic action of endo- and exo-glucanases that contribute to the occurrence of a peeling off mechanism. Cellulose accessibility analysis showed that limited accessible surface area of cellulose is probably not a major limiting factor that causes the decline of hydrolysis rate in its late stage. Restart hydrolysis experiment suggested that enzyme related factors such as enzyme inactivation or steric hindrance of enzymes should be responsible to the reduction in hydrolysis rate given the large size of cellulase enzymes.

June 21, 2016

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

James Liao Appointed To Serve As Academia Sinica President
Dr. James Liao has been named as the 11th president of Academia Sinica — the top academic research institution in Taiwan. Academia Sinica supports research activities in a wide variety of disciplines, ranging from mathematical and physical sciences, to life sciences and to humanities and social sciences. Dr. Liao serves as the BESC Activity Lead for Butanol Production by CBP.

June 20, 2016

Improved Thermophilic Anaerobe Achieves High Biofuel Titers From Wood And Cellulose
This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates. However,challenges were encountered with existing fungal cellulases and inhibitors from pretreatment. The high titers and yields observed support the feasibility of using engineered thermophiles for industrial ethanol production if challenges associated with pretreatment inhibitors can be avoided.

June 20, 2016

Relation between viscosity and shear rate of poplar lignin at 170 °C

Syringyl/Guaiacyl (S/G) Ratio In Lignin May Influence Quality Of Carbon Fiber Produced From Lignins
Current lignin-based carbon fiber using low-cost lignin precursors does not possess the mechanical properties required for many structural applications. Lignins with various syringyl/guaiacyl (S/G) ratios were isolated from Populus genotypes and subjected to rheological treatment at various temperatures and cooling conditions. Organosolv lignins extracted from genotypes with lower S/G ratios exhibited higher viscosity values and molecular weights with larger amounts of condensed structures following rheology treatment at relatively lower temperature; higher viscosity and molecular weight is known to provide for higher quality carbon fiber.

June 20, 2016

Isolation and characterization of M. truncatula mutant seeds lacking physical dormancy.

Discovery Of A Novel Molecular Mechanism Controlling Seed Physical Dormancy
Physical seed dormancy exists widely in higher plants, but its molecular mechanism has remained largely elusive. By screening a large number of Tnt1 retrotransposon-tagged Medicago truncatula lines, we identified non-dormant seed mutants from this model legume species. This study elucidated a novel molecular mechanism of physical dormancy and revealed a new role of class II KNOX genes.

June 07, 2016

Simultaneous Achievement Of High Ethanol Yield And Titer In Clostridium thermocellum
Adaptive evolution successfully improved the ethanol yield and the titer from a rationally engineered CBP strain. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.

June 01, 2016

Workshop Summary: Lessons From Industry In Commercializing Biotechnology
This article summarizes lessons learned drawn from the "Technology Challenges and Opportunities in Commercializing Industrial Biotechnology Workshop," September 28-29, 2015, San Diego.

May 09, 2016

A Bifunctional Ammonia-Lyase Is Critical In Grass Cell Wall Biosynthesis Of Lignin
Using phylogenetic, transcriptomic and in vitro biochemical analyses, a single homotetrameric cytosolic bifunctional ammonia-lyase (PTAL) was identified among eight BdPAL enzymes in the model grass species Brachypodium distachyon. 13C stable isotopic labelling experiments along with BdPTAL1-down-regulation in transgenic plants showed that the TAL activity of BdPTAL1 can provide nearly half of the total lignin deposited in grasses, with a significant preference for S-lignin and wall-bound coumarate biosynthesis.

April 29, 2016

Ball-milled whole cell wall poplar samples in different NMR solvents.

Improved Whole Cell Wall NMR Measures Carbohydrate And Lignin Structures Without Derivatization
A new NMR solvent system composed of dimethylsulfoxide (DMSO-d6) and hexamethylphosphoramide (HMPA-d18) was developed to understand plant cell wall structures with minimal deconstruction and modification of biomass. The proposed bi-solvent system does not require derivatization or chemical modification, thus it facilitates easier sample preparation and less signal overlapping of correlation peaks compared to the traditional methods.

April 28, 2016

BRC Directors

Intellectual Property Impact of the BRCs
The Bioenergy Science Centers (BRCs) have generated over 500 invention disclosures, 328 patent applications (of which 45 have been awarded) which have led to 133 licenses or options with industry.

April 28, 2016

Reduction of lignin content.

Identification And Overexpression Of Knotted1-Like Transcription Factor For Lignocellulosic Feedstock Improvement
Using transgenic approach, a switchgrass Knotted1 transcription factor, PvKN1 was overexpressed in switchgrass and transgenic plants were characterized for recalcitrance and growth-related traits. PvKN1 overexpression resulted in reduced lignin deposition/biomass recalcitrance and improved sugar release efficiency without affecting various growth parameters such as tiller height, tiller number, fresh and dry biomass.

April 26, 2016

QTL for lignin content and glucose release

New Genetic Markers Identified For Switchgrass Cell Wall Composition And Conversion
A full-sib switchgrass population was developed by crossing a lowland genotype (AP13) to an upland genotype (VS16). QTL associated with the traits of interest were identified. Significant variation for glucose, xylose and total sugar release, and lignin content (19-27%) in the population; a unit increase in lignin content reduced total sugar release by an average of 10 mg g-1; and QTL analysis detected nine genomic regions underlying sugar release and 14 for lignin content.

April 26, 2016

adhE expression and ethanol production with old vs. new plasmids

Development Of Improved Expression Plasmid In Clostridium thermocellum
Clostridium thermocellum is a promising candidate for ethanol production from cellulose biomass yet requires metabolic engineering to further improve ethanol yield. An expression plasmid was redesigned to improve transformation efficiency and structural stability. The new expression plasmid had both significantly higher transformation efficiency than its predecessor (~2x more), and was also able to more robustly express genes of interest.

April 26, 2016

Structural Diversity Of Xylans Measured In Cell Walls Of Monocots
This study provides a new perspective on the diversity, function and evolution of monocot cell walls, enabling the manipulation of xylan structure and development of new technologies for the conversion of biomass from this important family of plants to yield commercially valuable and sustainable products.

April 25, 2016

BRCs Well Represented at Symposium on Biotechnology for Fuels and Chemicals
The Symposium on Biotechnology for Fuels and Chemicals (SBFC) was held April 25-28, 2016, in Baltimore, Maryland. As part of the overall symposium the combined Bioenergy Research Centers (BRCs) presented thirty-seven talks and twenty posters representing 25% of the talks and 10% of the posters.

April 18, 2016

Photo of the field in June 2013.

Lignification And Recalcitrance Affected By Age And Season In Field-Grown COMT-Downregulated Switchgrass
Previous field and greenhouse studies have shown that COMT-downregulation in switchgrass decreases lignin content and reduces the S/G ratio, thereby lowering recalcitrance and improving ethanol yields. Trends in lignification and recalcitrance differ between growing seasons. Third-year results showed an increase in lignification and decrease in sugar release across the season. S/G ratios increased over time in non-transgenic control lines, but were relatively stable in COMT-downregulated lines. During early (elongation) growth stages, cell wall chemistry was similar between COMT-downregulated and control lines. After reaching the reproductive growth stage in July, COMT-downregulated lines maintained reduced lignin content, decreased S/G ratios, and improved sugar release through December. No differences in rust susceptibility were observed.

April 08, 2016

Dr. Willington Muchero, lead ORNL inventor

GreenWood Resources, Inc. Licenses BESC Invention For Lignin Regulator
Studies on the natural variation in lignin content in poplar trees identified a gene with major effects on lignin biosynthesis, the key polymer hindering efficient biomass digestibility. GreenWood Resources, Inc. has nonexclusively licensed this jointly-owned BESC invention and will continue to partner with ORNL as they complete field studies.

April 01, 2016

BioEnergy Science Center (BESC) Website Receives A "Distinguished Technical Communication" Award
The Washington-Baltimore Society for Technical Communication (STC) recognized the BioEnergy Science Center (BESC) website with the "Distinguished Technical Communication" award in April 2016.

March 30, 2016

Overall schemes of fuels from sunlight and atmospheric carbon.

Review: Using Microorganisms For The Production Of Next-Generation Biofuels
A review on how microorganisms can be exploited for the production of next-generation biofuels: a) based on the ability of bacteria and fungi to use lignocellulose; b) via direct CO2 conversion using microalgae; c) lithoautotrophs driven by solar electricity; or d) relying on the capacity of microorganisms to use methane generated from landfill.

February 05, 2016

TEM micrographs of Avicel particles after incubation with Clostridium thermocellum

Discovery Of New Enzyme System Sheds Further Light On High Cellulolytic Performance Of C. thermocellum
C. thermocellum utilizes not only all the cellulase degradation mechanisms known today (cellulosomes and free enzymes), but also a new category of scaffolded enzymes not attached to the cell. This discovery explains C. thermocellum's superior performance on biomass. These scaffoldins were found to be essential for the well known digestion mechanism, i.e., the defibrillation of plant cell walls.

February 04, 2016

CBP conversion of untreated Populus with high S/G ratio of lignin yielded significantly better hydrolysis and fermentation products.

Contribution Of Lignin Composition And Structure To Improvements In Bioprocessing Of Populus Using CBP
Populus variants with low and high syringyl to guaiacyl (S/G) ratios were hydrolyzed and fermented with Clostridium thermocellum under CBP conditions, and compared to conventional fungal enzymes and yeast. The composition and structure of cellulose and lignin in the biomass was correlated with the bioconversion results. Samples with a S/G ratio of 2:1 were converted more rapidly and to a greater extent than biomass with a 1:2 S/G ratio. The CBP microbe hydrolyzed biomass two to three times more readily than commercial fungal enzymes in either scenario. Lignin polymer size (lignin Mw) was found to be lower for the low S/G biomass.

January 12, 2016

Solubilization of washed mid-season switchgrass by various biocatalysts.

Optimizing Biocatalyst-Feedstock Combinations to Achieve High Solubilization with Minimal Pretreatment
This study carried out the most comprehensive controlled comparison to date of lignocellulosic solubilization by various biocatalysts, and report initial exploration of enhancing microbial solubilization of cellulosic biomass via mechanical disruption (cotreatment). Trends drawn from time-course and end-point data for six conversion systems and three substrates were: greater-than-expected differences in the effectiveness of various biocatalysts; lower yields and greater dependence on particle size for Populus as compared to green or senescent switchgrass; equal fractional solubilization of glucan and xylan with no biological solubilization of the non-carbohydrate fraction of biomass; and two-fold increase in solubilization via mechanical disruption with greater benefits on partially-fermented feedstock.

January 06, 2016

Hemicellulose and Lignin

Review: How Plant Cell Wall NMR Provides Insights Into Biomass Chemistry and Structures
The structure of the plant cell wall and recalcitrance are intimately tied together such that fundamental breakthroughs in recalcitrance have driven the science of biomass characterization. Work from BESC and other energy centers has dramatically enhanced the resolution and productivity of Nuclear Magnetic Resonance (NMR) analysis of biomass polymers including lignin, cellulose and hemicellulose. Whole cell NMR analysis, as reviewed in this article, provides a relatively facile means of characterizing changes in lignin and hemicellulose that contribute to reduced recalcitrance in pretreated biomass and/or transgenic or natural variant plant species.

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