Biomass Formation and Modification
Biomass Deconstruction and Conversion
Genome-Wide Association Study (GWAS) Dataset Released
The GWAS dataset comprises more than 28 million single nucleotide polymorphisms, or SNPs, derived from approximately 882 resequenced poplar genotypes. Each SNP represents a variation in a single DNA nucleotide, or building block, and can act as a biological marker, helping scientists locate genes associated with certain characteristics, conditions, or diseases. [Image courtesy ORNL]Read More
Natural Biocatalysts Outperform Industry Standard
Comparative study finds heat-loving microbes could reduce the need for thermochemical pretreatment
In the most comprehensive controlled comparison to date of lignocellulosic solubilization by various biocatalysts, BESC researchers found that naturally cellulolytic microbes such as Clostridium thermocellum (stained green and growing on poplar tissue in image) achieved significantly higher solubilization yields over commercial pretreatment systems. [Image courtesy ORNL]Read More
Promise of Unusual Lignin Polymer as Feedstock for Carbon Fiber
Stronger, Lighter Carbon Fiber Could Be Used in Parts for Cars, Aircraft, Electronics, and Sports Equipment
BESC researchers discovered an unusual lignin polymer in the seed coats of vanilla beans (pictured) that is naturally biosynthesized from caffeyl (C) alcohol. In contrast to lignin polymers that are highly cross-linked, C-lignin is a linear polymer, which makes it easier to process into high-quality carbon fibers. [Image courtesy iStock]Read More
Identification of Poplar Adaptation Mechanisms
Findings Could Lead to More Efficient Ways of Breeding High-Ethanol-Yielding Poplar and Other Tree Species
Using whole-genome sequencing and high-throughput phenotyping, BESC researchers were able to rapidly pinpoint gene mutations responsible for causing low lignin levels in poplar trees planted in a variety of environmental conditions. This proof-of-concept will allow researchers to tag genes associated with other economically important but complex plant traits. [Image courtesy BESC]Read More
Beneficial Plant Gene Discovery
Research Lays Groundwork for New Ways of Manipulating Plants
BESC researchers discovered a previously uncharacterized gene (GXMT1), which directs a key step in the development of xylan, a principal component of cell walls in plants such as Arabidopsis (pictured). Characterizing this gene, which makes the plant resistant to biofuel conversion, will lead to improved strategies for modifying such cell wall structures to help turn plants into biofuel more efficiently. [Image courtesy iStock]Read More
Molecular Structure of Plant Biomass
Imaging Reveals Enzymes in Action and Significance of Plants' Nanoscale Architecture
Plant cell walls contain networks of cellulose microfibrils that can be broken down into sugars for fuel and complex polymers that shield the cellulose from enzymatic attack. To characterize cell wall structure at the subnanometer scale and assess its response to chemical pretreatment, BESC developed techniques to image enzyme digestion in real time, revealing that biomass reactivity is determined by the nanoscale architecture of plant cell walls.Read More
A Leap Forward in Microbial Ethanol Yields
Engineered yeast converts up to 97% of plant sugars into fuel
Scientists at BESC and research partner Mascoma LLC have developed an advanced strain of Saccharomyces cerevisiae that sets a new standard for conversion of biomass sugars from pretreated corn stover. While conventional yeast (pictured) leaves more than one-third of plant sugars unused in the form of xylose, the C5 FUEL™ microbe efficiently converts this xylose into ethanol in less than 48 hours.Read More
Real-World Performance of Low-Lignin Switchgrass
Field study assesses sugar release and ethanol yield in transgenic feedstock
Lignin in the cell walls of switchgrass and other bioenergy feedstocks severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. In the first reported study of its kind, the biofuel potential of transgenic switchgrass with reduced lignin content was evaluated over two growing seasons.Read More
Using a "commercialization council" of technology transfer and intellectual property (IP) management professionals from partner institutions, BESC evaluates the commercial potential of new inventions arising from BESC research and promotes and facilitates the licensing of BESC IP.
BESC's education program, which is 75% self-sustaining, takes a novel approach. BESC has developed lesson plans to educate students about energy production and utilization. BESC uses a hub-and-spoke approach of working through regional science centers to maximize hands-on access and adaptation to local conditions.
Progress to Date
What are Biofuels? - Thanksgiving Point Gazette
June 19, 2017
Improving Bioavailability of Carbohydrate to C. bescii in Natural and Transgenic Switchgrasses
The bioavailability of carbohydrates to C. bescii was evaluated in the switchgrass lines unmodified Cave-in-Rock (CR), transgenic COMT-KD and Myb4-OE, and their corresponding parental lines. Each switchgrass line was examined as either hydrothermally treated (180ºC for 25 min) or untreated. C. bescii growth and carbohydrate solubilization (cellulose and hemicellulose) were determined for each treatment scheme (microbial, hydrothermal, or a combination of both). While C. bescii could significantly solubilize the transgenic switchgrass lines, including Cave-in-Rock, hydrothermal treatment was needed to solubilize 50% or more of the carbohydrate content.
May 01, 2017
Jonathan Mielenz, retired researcher from ORNL and BESC, was awarded the 2017 Charles D. Scott award (...more)
BRCs well represented at 39th Symposium on Biotechnology for Fuels and Chemicals
As part of the overall symposium the three DOE BRCs presented a combined total of twenty-two talks, including two as collaborations between the BRCs, and thirty-nine posters representing 28% of the talks and 15% of the posters. The presentations and posters highlighted the accomplishments, as well as common challenges in organization and research, in carrying out impactful mission-directed R&D in bioenergy.
May 01, 2017
New Insights On Recalcitrance From Lignin Inhibition Of Microbial Deconstruction
Microbial solubilization of biomass selectively targets carbohydrates. Clostridium thermocellum, a highly effective cellulolytic microbe, can hydrolyze ~93% of 100gL-1 pure cellulose. The microbe solubilizes only partially hydrolyzes cellulose in untreated energy crops. Techniques used were quantitative fluorescence colocalization analysis and surface chemical imaging. Quantitative evidence for the cause of feedstock recalcitrance: depletion of surface carbohydrate increases lignin exposure which leads to inhibition of enzymatic activity, while the bulk residual biomass retains significant undigested sugar content. Limited hydrolysis is not caused by inhibitory hydrolyzed sugars nor fermentation products (i.e., ethanol).
April 17, 2017
Photos of the field experiments in the first (2013), second (2014), and third (2015) growing seasons (...more)
Multi-Year Study Of Transgenic Switchgrass Plants In Regulation Shows Improved Yield And Dependency On Expression Levels
Four promising lines with moderate or low microRNA (miR156) overexpression from prior greenhouse study were selected for a field experiment to assess miR156 expression levels and biomass yields over three years. The best performing line consistently produced more biomass (25-56%) than the control in all three seasons, which translated to the production of 30% more biofuel per plant in the final season. Transgenic plants with modifications to transcriptional regulators, which impact such a broad range of genes, will likely require more extensive field evaluations than those with single and simple trait gene modifications to evaluate the stability of the desired growth phenotypes.
April 12, 2017
First-Of-A-Kind Study Of Thermophilic CBP With Cotreatment
Milling during lignocellulosic fermentation (or cotreatment) is an alternative to thermochemical pretreatment to enhance biological solubilization of lignocellulose. Carbohydrate solubilization was measured in consolidated bioprocessing with Clostridium thermocellum with cotreatment or with hydrothermal pretreatment. High carbohydrate solubilization was demonstrated without thermochemical pretreatment and added saccharolytic enzymes. C. thermocellum appears able to attack all the major linkages in cellulosic biomass provided that these linkages are accessible. The ability of C. thermocellum to withstand high intensity milling supports the feasibility of cotreatment. Less modified lignin may foster production of value-added coproducts.
March 27, 2017
Use of Caldicellulosiruptor saccharolyticus as a biological probe to report on changes in recalcitrance of plant biomass
C. saccharolyticus whole genome microarrays were used to access the transcriptome when grown on purified polysaccharides and chemically pretreated or genetically modified lignocellulosic substrates. Differential regulation of carbohydrate degradation, transport, and metabolism genes in C. saccharolyticus can be used to assess the availability of various plant polysaccharides to the microbe. This allows for inferences about the consequences of a chemical pretreatment or genetic modification of lignocellulose with an eye towards biofuels production.
March 21, 2017
Improving Pretreatment Inhibitor Tolerance in Clostridium thermocellum
Bioconversion performance in the presence of common pretreatment inhibitors was improved through targeted strain engineering thus reducing barriers to achieving a robust consolidated bioprocessing strategy with Clostridium thermocellum.
March 21, 2017
CBP-CT: an R&D-Driven Innovation with Potential for Disruptive Reductions in the Cost of Cellulosic Biofuels
Compared to current technology, the CBT-CT scenario was projected to offer 4.6-fold greater annual revenue, as well as 8-fold shorter payback period, and feasibility at ~10-fold smaller scale. CBT-CT is a potentially disruptive technology and a powerful, though still speculative, example of the potential benefits of including new-paradigm as well as in-paradigm innovation in R&D portfolios.
March 18, 2017
Overexpression of DUF266 can significantly enhance both cellulose and biomass in Populus
(BESC's 900th publication)
Little is known about the function of Domain of Unknown Function 266 (DUF266)-containing proteins and no DUF266 protein has been characterized in Populus. A bioinformatics approach was taken to identify all DUF266 proteins in Populus and their relationship with their homologs in other species. A transgenic approach was used to determine the function of one member of DUF266 proteins in Populus (PdDUF266A). The resulting phenotypes make PdDUF266A a promising target for genetic manipulation for improving biomass quality.
March 10, 2017
Use of Photoacoustic AFM to Spatially Resolve Nanomechanics of Plant Cell Wall Delignification
By chemically processing biomass and employing emerging nanometrology techniques, the various stages of lignin removal may be distinguished through the observation of morphochemical and nanomechanical variations. The cell wall nanomechanical properties undergo quantifiable reductions in plasticity, adhesion energy, and elasticity. These quantitative observations can be used to characterize delignification. The observed reduction in plasticity seems counterintuitive considering that lignin adds to cell wall rigidity.
February 27, 2017
Microbial Attachment to Cellulose Substrate Produces Widespread Gene Expression Changes
Unlike the majority of biofilm forming bacteria, C. thermocellum adheres to substrata that also provide its major carbon and energy sources. Before this study, discrete omics analyses of biofilm and planktonic cell populations had not been performed. Cellulolytic bacterial cells that evolved to thrive on solid carbon sources were shown to thoroughly alter expression of their central metabolism, anabolism, and homeostatic functions in response to the availability of solid attachment interfaces and solubilizable carbohydrates. The attached bacteria were functionally strong in growth and biomass conversion, while the planktonic cells were stressed and motile due to low available substrates.
February 07, 2017
Understanding the role of pentose sugars on C. thermocellum metabolism
This work highlights the importance of removing pentose sugars during bioconversion to biofuels, which could be achieved with C5 utilizing co-cultures or engineered strains of C. thermocellum, and is the first report of a functional ArgD-type cell signaling system in a thermophilic Firmicute and further work is warranted to understand the role of cell-to-cell signaling in achieving robust fermentations of lignocellulosic biomass.
January 20, 2017
Visualization of Significant Alteration of Populus biomass by C. bescii
Caldicellulosiruptor bescii targets surface cellulose and hemicellulose, but increase of surface lignin could inhibit further sugar access. Biphasic cell growth is attributed to degradation of easily accessible sugars followed by utilization of more insoluble polysaccharides.
January 10, 2017
Metabolic Adaptation Of C. thermocellum To Growth Inhibitors Released During Deconstruction Of Switchgrass
Integrated omics data of microbial growth on complex lignocellulosic biomass over time provided a detailed view of the molecular machinery (metabolites and enzymes) that reveals temporal adaptation to a complex, lignocellulose substrate — information that is critical for engineering C. thermocellum's industrial efficacy.
January 03, 2017
Engineering N-terminal End of CelA Enhances the Cellulolytic Activity of Caldicellulosiruptor bescii
To test whether alteration of the N-terminal terminus of CelA GH9 and CelA GH48 domains might improve secretion and/or catalytic efficiency of CelA, repeating aspartate tags were introduced into the N-terminal ends of these 8 domains. Introduction of repeating aspartate tags resulted in an increase in the general activity of the exoproteome and a dramatic increase in growth of C. bescii on crystalline cellulose. Most efficient cellulase systems contain highly active exocellulase enzymes capable of decrystallizing cellulose, so the observed increase in activity on Avicel bears directly upon the ability of CelA to degrade realistic biomass feedstocks destined for biofuels production.
December 22, 2016
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 22, 2016
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 12, 2016
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
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
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
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
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.