"The initial vision of BESC was to
understand and overcome recalcitrance
in plants, since this will lead to
outcomes that impact the economics
of biofuel production from lignocellulosic
materials. Significant advances
toward that goal have positioned
BESC to directly address many underlying
key cost factors in the overall
process. From engineering microbes
that consolidate multiple processes to
modifying plants that exhibit reduced
recalcitrance, the BESC team has moved the boundaries of
cellulosic biofuel science to reveal insights that in turn have led
to these achievements. Furthermore, BESC has demonstrated
how to successfully run an organization whose collaborators
are highly distributed both geographically and by scientific
discipline. Based on the extensive body of scientific knowledge
that BESC scientists have contributed to the field, the
center's goals now are to go well beyond proof of principle and
deliver functional developments to the biofuels
– Paul Gilna
Paul Gilna is BESC's director and deputy director of Oak Ridge National Laboratory's Biosciences Division. With a multidisciplinary background in pharmacology, bioinformatics, computational biology, and microbial genomics, Gilna has led other large collaborative bioscience projects, including ones associated with GenBank, the DOE Joint Genome Institute, and CAMERA (Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis). He also has served as a program director at the National Science Foundation.
Biomass recalcitrance—the resistance of plants to deconstruction—is the primary barrier to efficiently and economically accessing fermentable sugars for advanced biofuels that will directly displace petroleum. Understanding and overcoming this recalcitrance are central research themes of the DOE BioEnergy Science Center (BESC) led by Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee. BESC's defining goal is to enable the emergence of a sustainable cellulosic biofuels industry by leading advancements in science and science-based innovation resulting in removal of recalcitrance as an economic barrier. Convinced that biotechnological approaches hold the most promise for achieving these breakthroughs, BESC is developing plants that are easier to deconstruct and microbes that more effectively convert lignocellulose into simple sugars. When established by DOE in 2007, BESC sought researchers from institutions across the United States to bring breadth and depth of expertise to the challenge of overcoming biomass recalcitrance.
Transformative advances in understanding recalcitrance require detailed knowledge of the chemical and physical properties of biomass that influence its resistance to degradation. Research has been aimed at determining (1) how these properties can be altered by engineering plant biosynthetic pathways, (2) how biomass properties change during pretreatment, and (3) how such changes affect biomassbiocatalyst interactions during deconstruction by enzymes and microorganisms. Historically, the term "recalcitrance" was coined to describe an overall phenotypic trait of biomass, namely the degree of difficulty in obtaining access to sugars complexed in the plant cell wall. However, based on new knowledge about cell wall chemistry, structure, and biochemistry, BESC researchers have redefined recalcitrance as a phenomenon in terms of pathways and interactions, both in cell wall formation and bioconversion. This increasing knowledge of the scientific basis of recalcitrance underpins the overall BESC goal of eliminating it as an economic barrier to cost-effective biofuel production.
In the past 6 years, BESC has made crucial progress toward understanding, manipulating, and managing plant cell wall recalcitrance and conversion. Notably, the BESC team proved the core concept that multiple genes control cell wall recalcitrance and that manipulating these genes potentially could yield perennial biofeedstocks that are easier to deconstruct. This research paves the way for improving feedstocks directly or by genetically assisted breeding. In conversion science, BESC researchers have identified and validated key genes for consolidated bioprocessing (CBP), a game-changing, one-step strategy that uses a single microbe or microbial consortium to both deconstruct biomass and ferment resulting sugars into fuels. Researchers are beginning to modify CBP target organisms to improve conversion and enhance products. In addition, they have shown the potential of thermophilic (heatloving) microbes in biomass conversion and identified the critical deconstruction enzymes for key components of lignocellulosic biomass. Currently, the BESC team is demonstrating the action of improved CBP on modified plant cell walls.
BESC is organized into three research focus areas: (1) Biomass Formation and Modification, (2) Biomass Deconstruction and Conversion, and (3) Enabling Technologies—all supported by integrating activities. Some recent highlights of BESC research are featured below, and all BESC publications are available for download from the center's website, bioenergycenter.org.
As part of its goal to reduce biomass recalcitrance, BESC conducts molecularlevel investigations of cell wall assembly and polysaccharide and lignin synthesis to gain an in-depth mechanistic understanding of recalcitrance. Scientists also use targeted modification of plant cell walls and selection of natural feedstock variants to reduce recalcitrance and improve biomass characteristics. Although these methods can be applied to a wide range of woody and herbaceous plants, BESC focuses on two potential bioenergy crops, poplar and switchgrass, with improved characteristics for converting cellulosic biomass to fermentable sugars. Researchers are moving promising plants with improved traits from greenhouses into field trials where their real-world usefulness can be evaluated. Successfully reducing recalcitrance will greatly facilitate the efficiency and economic viability of advanced biofuels.
Two key hypotheses drive Biomass Deconstruction and Conversion research at BESC: (1) microorganisms can be engineered to enable CBP and (2) enzymes and microbial biocatalysts can be understood and engineered to synergize with recalcitrance-reducing plant modifications to achieve better biomass deconstruction.
To test these hypotheses, BESC targets three microbial CBP platforms: yeast, Clostridium thermocellum, and Caldicellulosiruptor species. In yeast, a robust industrial ethanol producer, researchers have pursued genome modifications aimed at improving the expression and activity of enzymes that will digest cellulose. Already natural cellulose degraders, the other two microbes are thermophilic anaerobes whose metabolism is being manipulated by BESC to produce useful products such as ethanol or butanol.
Studies of microbial cellulose utilization at all levels—including enzyme components, enzyme systems, pure cultures, and mixed cultures—demonstrate the increased effectiveness of microbial cultures compared with free enzymes acting in the absence of cells. Enabling CBP will reduce the need for pretreatment chemicals toxic to microbes and will reduce the energy required to deconstruct and convert biomass into biofuels.
Optimizing Microbes for Biomass Deconstruction and Conversion. Kelsey Yee operates a processcontrolled Applikon fermenter to evaluate how well Caldicellulosiruptor obsidiansis (a consolidated bioprocessing microbe) ferments simple sugars derived from poplar pretreated with dilute acid. [Image courtesy of Oak Ridge National Laboratory]
Analyzing Biomass Recalcitrance. Steve Decker watches a robot dispense samples of powdered biomass into a reactor plate as part of a high-throughput recalcitrance pipeline for studying sugar release in potential biofuel feedstocks. [Image courtesy National Renewable Energy Laboratory]
BESC characterization and computational modeling efforts are focused on developing and applying chemical, immunological, physical, and imaging methods to characterize biomass. The resulting information is then used to identify relationships between biomass structure and recalcitrance.
BESC has developed a suite of new enabling technologies that support and enhance feedstock and CBP research. These technologies include high-throughput (HTP) assays for determining the recalcitrance properties of tens of thousands of feedstock samples. The information obtained from HTP pipelines for pretreatment, digestibility, and chemistry has enabled plant scientists to discover improved plant lines and single-nucleotide polymorphisms (SNPs) that link genotypic variation to cell wall chemistry in natural populations of poplar and switchgrass.
Tests that integrate enhanced-biomass grasses with cellulolytic microorganisms are showing greater effectiveness in conversion and biofuel production, supporting a BESC hypothesis that minimal to no pretreatment may be possible for some conversion regimes.
Modeling and simulation tools developed by BESC researchers are being used to analyze various aspects of recalcitrance. Flux-balance analyses have provided insight into lignin synthesis, and several tools are publicly available within the BESC KnowledgeBase to analyze, for example, carbohydrate-active enzymes (CAZymes) and regulons.
These enabling technologies not only have advanced the science of biomass production more quickly, but also can be applied to numerous other areas of biological research.
Translating BESC research results into the testing of applications and potential commercial deployment is an important step toward reaching DOE's bioenergy objectives. 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. A searchable BESC invention website is located under the Industry tab on the BESC home page (bioenergycenter.org), providing easy access to BESC IP available for licensing. Along with BESC researchers, industrial partners and members of the commercialization council attend and actively participate in the center's annual retreat.
Results from BESC's Biomass Deconstruction and Conversion research are being tested and applied by industrial partners Mascoma Corporation and DuPont. BESC works with Mascoma to test on a pilot scale the first CBP yeast strains able to express multiple hydrolytic enzymes while remaining robust enough to produce ethanol with little to no added commercial enzymes. Other yeast strains are studied at various levels—including pathways, enzymes, CAZymes, protein domains, SNPs, and indels—to identify functionally significant variations. BESC partners with DuPont to test improved plant lines in a bench-top simulation of the company's commercial process for lignocellulosic biofuel production.
Other direct interactions with industry include partnering with Ceres, ArborGen, and GreenWood Resources, Inc., to evaluate improved bioenergy feedstocks. For example, Ceres and BESC are conducting field trials of modified switchgrass lines. Preliminary results from these trials, which are approved by the U.S. Department of Agriculture, closely follow those from greenhouse and laboratory studies. For field testing of woody feedstocks, ArborGen generates stable poplar transgenic species, including lines with altered cell wall composition. GreenWood Resources operates one of the field sites in a study examining 1,000 natural poplar variants now in its fourth year.
In addition to these activities, BESC recently initiated a webinar series to inform interested industry professionals of BESC science. In the first webinar, BESC Director Paul Gilna provided an overview of the center. The series continued with webinars by BESC researchers Steve Brown and Neal Stewart highlighting, respectively, BESC microbial and "omics" capabilities and switchgrass field trials at the University of Tennessee.
To prepare the next generation of bioenergy scientists, BESC provides interdisciplinary research opportunities to graduate students, postdocs, and visiting scientists. Hands-on "Farming for Fuel" lessons educate students in the fourth–sixth grades about a bio-based fuel economy. Working with the Creative Discovery Museum in Chattanooga, Tennessee, BESC has reached 100,000 (~20,000 in year 7 alone) students, teachers, and parents nationwide. A free iPad app featuring a Biofuel Road Trip Challenge is available for download under the education category on iTunes. Materials and lessons are available at bioenergycenter.org/besc/education.cfm.
BESC researchers are building and applying imaging technologies and platforms to characterize the structure of plant biomass at the molecular level and assess how it is affected by chemical pretreatment. A BESC-developed integrated microscopy system combining atomic force microscopy, stimulated Raman scattering microscopy, and single-molecule spectroscopy was used for subnanometer imaging and quantitative chemical mapping of pretreatment and enzyme digestion in real time. With this new technology, researchers were able to localize the enzymatic sites of action without compromising the cell wall's structural integrity. The study revealed that biomass reactivity is determined by the nanoscale architecture of plant cell walls. Lignin, a major component of this architecture, physically impedes the accessibility of chemical and enzymatic catalysts to substrates. Results suggest that biomass pretreatment ideally should focus on eliminating lignin while leaving intact the structural polysaccharides within cell walls. Such pretreatment would result in a structure that allows easy access by enzymes and rapid digestion of polysaccharides, leading to more efficient and cost-effective biofuel production processes (Ding et al. 2012).
BESC scientists also have devised new imaging methods to study biomass-biocatalyst interactions. ORNL researchers at BESC received an R&D 100 Award for developing a modesynthesizing atomic force microscope that allows molecular-level spectroscopic measurements of plant tissues at 50 nm resolution (Tetard, Passian, and Thundat 2010). Other advanced imaging methods have been applied to analyze, for example, surface biomass using time-of-flight secondary ion mass spectrometry/matrixassisted laser desorption and ionization mass spectrometry ( Jung et al. 2012), as well as cell wall lignin and cellulose using a coherent Raman scattering technique. In addition, BESC researchers have used total internal reflection fluorescence microscopy to image how enzymes interact with plant polysaccharides and have obtained nanometer-scale images of hydrated plant cells, which identified key parameters in reducing the recalcitrance of transgenic plants (Liu, Ding, and Himmel 2012).
Previously, the majority of genes responsible for plant cell wall recalcitrance were thought to be located in the metabolic pathways responsible for lignin synthesis. BESC's early work showed that recalcitrance, and thus biofuel yields, could be improved by manipulating genes in these pathways. However, in recent years, BESC researchers have demonstrated that additional cell wall biosynthesis genes outside the lignin pathway have significant effects on recalcitrance. These include:
Because higher temperatures facilitate the deconstruction of lignin and release of simple sugars within plant biomass, thermophilic bacteria are promising candidates for biofuel production systems. The thermophiles Clostridium thermocellum and Caldicellulosiruptor bescii, in particular, hold great potential for consolidated bioprocessing (CBP), a strategy that uses a single microbe or culture to both deconstruct biomass and ferment resulting sugars into fuels. To take full advantage of this potential, BESC researchers are using new genetic tools to create CBP production strains of these bacteria with high yield and conversion. In C. thermocellum, researchers conducted various manipulations of the microbe's carbon, electron, and ethanol tolerance pathways. The most successful strategy to date improved ethanol yield to nearly a third of a gram of fuel per gram of carbohydrate (Deng et al. 2013). This yield represents more than a threefold increase over the wild type strain and is a significant step toward a theoretical goal of half a gram of fuel per gram of carbohydrate.
BESC scientists also have developed the first system allowing stable introduction of foreign DNA elements into C. bescii, which produces primarily lactate, acetate, and hydrogen as fermentation products. This breakthrough is based on identification of a Caldicellulosiruptor "immune system" that normally protects the bacterium from viral infection, destroying outside DNA before it can be integrated into the host genome. The BESC team developed a set of targeted nucleic acid modifications that protects DNA from the host immune system, allowing introduction of new genes and regulatory elements into the organism. This technology will enable metabolic engineering of C. bescii for direct conversion of lignocellulose to biofuels such as ethanol and butanol (Chung et al. 2012).
In related research, BESC demonstrated the first targeted gene deletion for this microbe— the gene encoding lactate dehydrogenase (ldh). The deletion was constructed within a nonreplicating plasmid (a small DNA molecule physically separate from chromosomal DNA) and then introduced into the C. bescii chromosome. Because the plasmid contains a gene for which there is both positive and negative selection, researchers were able to select first for recombination of the deleted ldh gene and then for loss of the plasmid sequences. Because this method allows for clean genetic insertions and deletions that leave no residual genetic material, it can be used repeatedly for metabolic engineering. The C. bescii strain containing the ldh gene deletion exhibited the expected changes in metabolism, namely the inability to produce lactate, leading to increased acetate and hydrogen production. This demonstration of a gene replacement strategy paves the way for further genetic manipulation of C. bescii to produce desired biofuel fermentation products directly from plant biomass. Future goals target increased yield and titer as well as improved understanding of cellulolytic fermentation, enzymes, and regulation (Cha et al. 2013).
In addition to their natural ability to break down lignocellulose, C. thermocellum and C. bescii have the surprising capacity to extensively deconstruct biomass (especially grasses) after minimal or no chemical pretreatment, a typically harsh and expensive step in biofuel production. To understand the mechanisms underlying these abilities, BESC researchers compared two enzyme systems for degrading biomass: "cell-free" fungal cellulases and cellulosomes—large multiprotein complexes that protrude from a bacterium's surface, attaching to and digesting plant cell walls. The BESC team demonstrated that solubilization of the cell wall is far more effective when mediated by cellulolytic microbes (e.g., C. thermocellum and C. bescii) than by cell-free enzymes. Provisional patents were filed on the discoveries that CelA from C. bescii is the most active enzyme reported to date on Avicel cellulose and that C. thermocellum cellulosomes will enhance the activity of fungal hydrolytic enzyme cocktails. Improved understanding of the mechanistic differences between these two enzyme systems highlights new opportunities for combining them to enhance cellulose degradation and sugar release in biofuel production processes (Resch et al. 2013).
BESC researchers also have shown that C. bescii can simultaneously solubilize lignin, cellulose, and hemicellulose from switchgrass at high temperatures (78°C) without chemical pretreatment. Until now, no anaerobic microbe had been known to degrade lignin. Although C. bescii did not consume lignin, it removed it, thereby improving access to the plant's energy-rich sugars. The discovery of a single microbe that can efficiently grow on plant biomass, degrade it, and biosynthesize fermentation products offers the potential for streamlining biofuel production (Kataeva et al. 2013).
When BESC was formed, methodologies for the genetic transformation of switchgrass (Panicum virgatum L.) were relatively inefficient. Over the last 6 years, BESC has created a rapid, stable transformation protocol for switchgrass that has now been standardized across the center. This protocol has improved the transformation efficiency from 20% to >90% and decreased the turnaround time for generation of new transformants from months to weeks (Wang 2013, unpublished; Nageswara-Rao et al. 2013).
To support gene discovery and analysis in switchgrass, BESC researchers also developed an integrated transcript sequence database and a gene expression atlas containing quantitative transcript data for most genes in all the major switchgrass organs. Researchers assembled full-length or partial mRNA sequences for a majority of the plant's functional genes and developed a web server (switchgrassgenomics.noble.org) for hosting all switchgrass datasets. This site serves as a robust tool for functional genomics and breeding in switchgrass, providing annotations that will help users formulate hypotheses about gene function. Because the server also handles RNA-seq data, it can act as a "one-stop" shop for switchgrass transcriptomics (Zhang et al. 2013).
A new study that uses glycome profiling to monitor biomass changes in structure and sugar extractability during
pretreatment suggests that lignin content per se does not affect recalcitrance. Instead, it is the integration of lignin and
polysaccharides within cell walls—and their associations with one another—that appear to play a larger role. To better
understand how the pretreatment process reduces plant cell wall recalcitrance, researchers adapted glycome profiling
protocols to develop a medium-throughput technique to analyze poplar samples from BESC and stover samples from the
Great Lakes Bioenergy Research Center. Glycome profiling uses polysaccharide antibodies to identify changes in plant cell
wall polysaccharide epitopes. Results from this study demonstrated the sequence of structural changes that occur in plant
cell walls during pretreatment-induced deconstruction, namely, the initial disruption of lignin-polysaccharide interactions
in concert with a loss of pectins and arabinogalactans; this is followed by significant removal of xylans and xyloglucans
(Pattathil et al. 2012).
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