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Genomic Science Program

DOE Great Lakes Bioenergy Science Center (GLBRC)

Donahue

"GLBRC researchers, in partnership with the state of Wisconsin, the state of Michigan, and affiliated industries, have made substantial progress toward developing the next generation of advanced biofuels. We are committed to building on these scientific breakthroughs and accelerating our efforts to develop sustainable biofuel strategies, from growing plants for use as energy feedstocks to exploring novel ways to convert the nonedible components of plants into fuels for the automotive, diesel, and aviation sectors." – Tim Donohue

Tim Donohue is GLBRC's principal investigator and director, as well as a professor of bacteriology at the University of Wisconsin–Madison. He is an expert in applying the latest genomic and systems biology approaches to understanding how genetic pathways and networks in microorganisms are used to generate cell biomass or biofuels from sunlight.

The DOE Great Lakes Bioenergy Research Center (GLBRC) is led by the University of Wisconsin– Madison, in close partnership with Michigan State University. Located in the world's most productive agricultural region, the GLBRC is exploring scientifically diverse approaches to converting sunlight and various plant feedstocks—agricultural residues, wood chips, and grasses—into biofuels.

The center supports nearly 400 researchers, students, and staff spanning a wide array of disciplines, from microbiology and plant biology to engineering and economics. The innovations born of these unique collaborations provide the basic scientific foundation for the sustainable, large-scale production of advanced cellulosic biofuel technologies that will help meet the nation's growing energy needs.

Taking full advantage of the diverse expertise of its research team, GLBRC leadership is focusing on two key knowledge gaps: sustainable production of crops with desirable biofuel traits and efficient conversion of biomass into fuels and chemicals. The economic and environmental sustainability of cellulosic technologies depends greatly on how biofuel crops are produced and whether they compete with food production for land use. Optimal sustainable biofuel crops will have different traits than those of crops used solely for food. Alternatives to cellulosic ethanol—referred to as advanced biofuels—also hold promise for the automotive, diesel, and aviation sectors. The GLBRC research portfolio is strategically positioned to increase emphasis on production of advanced biofuels and to modify deconstruction and conversion technologies to maximize the efficiency of lignocellulosic biomass processing.

As the center has matured, it has increased its focus on areas that the biofuels industry categorizes as obstacles to the sustainable, efficient, and cost-effective production of energydense fuels and chemicals from lignocellulosic biomass. One such obstacle is lignin, and GLBRC researchers have made important advances that will reduce the energy needed to use this promising source of fuels and chemicals. By applying unique alkaline pretreatment strategies, the GLBRC hopes to harness a reliable lignin stream that will open up tremendous possibilities for coproducts and materials that can add value to the biofuels pipeline.

From an organismal perspective, researchers are closely examining the stressors that keep microbes from doing their jobs effectively. Going forward, the GLBRC will continue using genomic tools to discover insights that will enable microbes to handle those stressors. From a broader perspective, GLBRC sustainability researchers have been concerned since the center's inception about how to introduce bioenergy crops into the landscape without causing unintended environmental, economic, or social consequences. One possible and promising solution is the use of marginal lands.

 

Research Focus Areas

With a focus on sustainable biofuel production as an organizing principle, the GLBRC science portfolio is built upon four integrated research areas: Sustainability, Plants, Deconstruction, and Conversion. Research support activities within the center enable faster, more collaborative research and engage scientists in training future bioenergy leaders.


1. Sustainability

For the bioenergy economy to have a positive impact on the United States, complex issues in agricultural, industrial, and behavioral systems must be addressed. To determine the best practices for biofuel production, GLBRC researchers study issues such as minimizing energy and chemical inputs for bioenergy crop production; reducing greenhouse gas emissions from the entire biofuel production life cycle; and understanding the environmental impacts of removing leftover stalks, stems, and leaves from food crops. GLBRC scientists also study the social and financial incentives needed to promote the adoption of more environmentally beneficial practices.

hydrolysate

Improving Biomass Conversion. Hydrolysate is a sludgy mix of partially digested plant material resulting from the enzymatic hydrolysis of biomass. During fermentation, microbes encounter sugars in hydrolysate plus a variety of other compounds, some of which may slow their ability to create fuel. GLBRC researchers are studying ways to make this process more efficient. [Image courtesy GLBRC]

2. Plants

With the goal of improving the traits and sustainable production of bioenergy crops, GLBRC researchers are investigating how genes affect cell wall digestibility in model plants, cornstalks, and switchgrass. A key breakthrough in bioenergy feedstock development has been the engineering of new forms of lignin that promote biomass digestibility. Modified lignin molecules can significantly decrease the time and energy required for biomass processing by making recalcitrant plant tissues easier to break down and convert to fuels. Additionally, GLBRC researchers are breeding plants that produce more hemicelluloses and oils that can be converted into biofuels. They are increasing the energy density of grasses and other nontraditional oil crops by manipulating the metabolic and genetic circuits that control accumulation of oils and other easily digestible, energy-rich compounds in plant tissues.

3. Deconstruction

Located at the intersection of America's agricultural heartland and its northern forest, the GLBRC has access to a rich diversity of raw biomass for study. GLBRC Deconstruction researchers are discovering and improving natural cellulose-degrading enzymes extracted from diverse environments. Improved enzymes created by the GLBRC protein-production pipeline are used in analyzing a range of plant materials and pretreatment conditions to identify the best combination of enzymes, chemicals, and physical processing for enhancing the digestibility of specific biomass sources. Deconstruction researchers also are exploring ways to add value to these processes by developing pretreatment technologies that can yield additional compounds and coproducts useful for energy applications.

4. Conversion

The need for increased quantity, diversity, and efficiency of biomass-derived energy drives GLBRC Conversion research. Along with continuing to develop cellulosic ethanol processes, the center is moving forward with a focus on improving biological and chemical methods for converting plant material into advanced biofuels and chemicals that can replace fossil fuels.

Research Support

Crossing all research areas, GLBRC's Core Facilities area provides cutting-edge technologies that facilitate the innovative discoveries and creative solutions needed to advance bioenergy research. These facilities provide specialized highthroughput screening for plant cell wall digestibility and chemical composition, plant transformation, proteomics, and metabolomics. This research support area provides core plant transformation facilities for Arabidopsis, Brachypodium, and maize. It also operates the hydrolysate production chain that uses quality-controlled methodologies to generate biomass hydrolysates for fermentation experiments.

The Informatics and Information Technology (IIT) team develops and delivers optimal computational solutions and ensures their effective adoption in support of the GLBRC's overall mission. IIT focuses on the delivery and management of a laboratory information management system and the creation of tools and methodologies for collaboration and knowledge management among GLBRC staff. The team also works closely with center scientists to support experimental design, data analysis, and interpretation, enabling practical and conceptual breakthroughs in biofuels production.

Education and Outreach

The staff and partners of the GLBRC Education and Outreach team inform a variety of audiences about biofuels research, energy concerns, and sustainability issues affecting our planet. The team's goal is to broaden the understanding of current bioenergy issues for the general public, as well as students and educators at K–12, undergraduate, and graduate levels. In the GLBRC's suite of bioenergy education materials for teachers and students, a strong emphasis is placed on using critical thinking, quantitative reasoning, and systems-based logic. Because bioenergy research and development are important contemporary issues, Education and Outreach members present GLBRC research to diverse audiences at numerous events and programs in an accessible and interesting way.

Industry Partnerships

The GLBRC uses a fundamental, systems-driven, and genomeinformed basic science approach within a project management environment and thus operates primarily in the early research and development arena. Once technology is developed, the center works closely with industry partners—through technology transfer mechanisms or collaboratively—to achieve commercial implementation.

GLBRC intellectual property is protected by the Wisconsin Alumni Research Foundation (WARF), which was established in 1925 as the world's first university-based technology transfer office. WARF also supports GLBRC innovation by conducting intellectual property surveys to guide new research and by licensing discoveries to companies for commercial uses that benefit society. With assistance from WARF, GLBRC researchers will begin scaling up a process that uses a recyclable organic solvent called gamma-valerolactone to generate high yields of sugars from cellulosic biomass. The sugars produced from this process can then be chemically or biologically upgraded into advanced biofuels.

The first patent and license on GLBRC technologies were both issued in 2012, with many more expected in the next 5 years. The center has submitted 66 patent applications, 82 invention disclosures, and 23 licenses since its inception in 2007.

As one of the GLBRC's primary industry partners, the Michigan Biotechnology Institute (MBI), a subsidiary of the Michigan State University Foundation, has worked with research area leaders to lower the commercial risk of potential biofuel technologies. In addition, the private company Hyrax Energy, Inc., was founded in 2011 based on GLBRC research. As the first company to emerge from GLBRC research, Hyrax also secured the first license on a GLBRC technology.

GLBRC Partners

DOE BRC Partners

Map of DOE Bioenergy Research Centers and Partners [Image courtesy ORNL]

  • University of Wisconsin–Madison (lead institution): The GLBRC's lead partner provides world-renowned expertise in genome-enabled analysis of plant and microbial pathways, networks, and systems; computational analysis of bioenergy proteins, organisms, and ecosystems; and discovery, production, and improvement of bioenergy enzymes.
  • Michigan State University (MSU): MSU researchers are experts in the breakdown and synthesis of plant cell walls, oils, and other polymers; the breakdown of cellulose in plant stems, stalks, and leaves, including trees and other woody plants; and the development of environmentally and economically sustainable biofuel production practices.
  • Illinois State University: Researchers at Illinois State work on the genetic and molecular analysis of switchgrass.
  • Lucigen Corporation: Lucigen provides valuable expertise in bioprospecting for new biomass deconstruction enzymes.
  • DOE's Pacific Northwest National Laboratory (PNNL): PNNL enables the GLBRC to analyze the entire life cycle of bioenergy practices.
  • University of British Columbia (UBC): Work at UBC contributes additional lignin expertise to the GLBRC's Plants area, including plant cell wall lignification and cross-linking to modify structure and improve biomass processing.
  • University of New Hampshire (UNH): GLBRC Sustainability researchers at UNH investigate how bioenergy crops influence the composition and enzymatic activity of soil organic matter.
  • University of Toledo: Within the GLBRC's Sustainability area, experts in environmental studies work to understand the biogeochemical cycling of bioenergy crop production.
  • Texas A&M University: As part of the Plants area, Texas A&M researchers study sorghum as a source of stem sugars for *See back cover for map showing partner locations. energy production.

Research Highlights

Examining the Capacity of Marginal Lands to Support Biorefineries

An issue concerning cellulosic biofuel systems is the use of land for bioenergy feedstocks versus food crops. Marginal lands offer a potential solution because they are unsuitable for growing food crops but can support grasses and other plants with bioenergy potential. Using 20 years' worth of long-term ecological research data from 10 midwestern states, GLBRC Sustainability researchers found that mixed-species plants from marginal lands produce as much biomass as traditional feedstocks such as corn grain. Moreover, these plants reaped twice the climate benefits of corn by sequestering more carbon in the soil and reducing fossil fuel consumption. Out of nearly 27 million acres of estimated marginal land, researchers found 35 locations in the 10 states that could each support a biorefinery with a capacity of at least 24 million gallons of ethanol per year. Including these sites, researchers identified enough marginal land in the study area to produce 5.5 billion gallons of ethanol per year, amounting to 25% of the mandated 2022 target for cellulosic biofuels. The results suggest that using marginal lands for bioenergy feedstocks would eliminate land-use competition between food production and biofuels (Gelfand et al. 2013).

Maximizing Switchgrass Productivity

GLBRC Plants researchers are developing resources and tools to improve switchgrass varieties by breeding this potential biofuel feedstock for climates similar to the midwestern and northern United States. In particular, researchers are applying genomic selection to plant breeding and developing a hybrid production system to maximize switchgrass productivity. Combining genomic selection, late-flowering winter-hardy genotypes, and hybrid deployment has the potential to double switchgrass yields by 2020 (Casler 2010).

Enhancing bioenergy feedstocks

Enhancing Bioenergy Feedstocks. GLBRC technician Nick Baker and researcher Rajan Sekhon harvest young switchgrass tissue for RNA and DNA analysis. [Image courtesy GLBRC]

Characterizing Switchgrass Diversity

In collaboration with the DOE Joint Genome Institute ( JGI), BioEnergy Science Center (BESC), Joint BioEnergy Institute ( JBEI), and others, GLBRC Plants researchers are enhancing resources that can characterize the natural diversity in switchgrass, including its reference genome sequence. This sequencing will characterize the genetic variation of structured populations and diverse genotypes of broad interest to the switchgrass community and will facilitate high-density genotyping necessary for switchgrass genomic selection, gene discovery, and evolutionary studies. These enhanced genetic resources will accelerate the breeding of switchgrass for use as a biomass crop (Zhang et al. 2011a).

Biomass-Deconstructing Enzymes from Wasp Communities

Biomass deconstruction is a major step in the cellulosic biofuels pipeline, and GLBRC scientists have looked to natural environments for solutions to breaking down cellulose. For example, Deconstruction researchers have isolated a new aerobic microbe called Streptomyces sp. ActE from a community of wood-boring wasps. ActE may aid development of specialized approaches to deconstructing woody materials because of an apparent evolutionary advantage enabling it to efficiently break down cellulose. Researchers used genome-wide transcriptomic and proteomic analyses to identify the suite of enzymes used to deconstruct crystalline cellulose and other pure polysaccharides that ActE secretes when grown on plant biomass. The mixture of enzymes obtained has biomass-degrading activity comparable to a cellulolytic enzyme cocktail from the fungus Trichoderma reesei. This example of high cellulolytic capacity in an aerobic bacterium is novel to biofuels research, and identification of these new enzymes greatly expands the repertoire available for biomass deconstruction (Takasuka et al. 2013). Accelerating

AFEX

GLBRC Pretreatment Technology. AFEX™-treated corn stover with water before enzymatic hydrolysis, a process that breaks down cellulose polymers into simple sugars using cellulase enzymes. [Image courtesy GLBRC]

Accelerating Ethanol Production with Novel Yeast Strains

GLBRC Deconstruction and Conversion researchers have improved the efficiency of cellulosic biomass enzymatic hydrolysis and fermentation, decreasing the total time for ethanol production from over a week to two days. Using ammonia fiber expansion (AFEX™), a biomass pretreatment technology, Deconstruction researchers developed the simultaneous saccharification and cofermentation process (SSCF) to combine enzymatic hydrolysis and fermentation of corn stover into a single reactor. Conversion researchers subsequently performed phenotypic screening and engineering of yeast to develop a strain with increased tolerance to the heat and degradation products present in SSCF reactions, as well as the ability to metabolize xylose. The collaboration resulted in a novel yeast strain able to thrive in the unique SSCF environment and produce ethanol concentrations comparable to a welldeveloped industrial benchmark strain ( Jin et al. 2013).

Adding Value to Biomass Processing with Lignin

GLBRC researchers are using highly selective catalysts to oxidize lignin molecules for conversion into valuable chemical feedstocks that potentially could replace petroleum-based compounds. Exemplifying the center's emphasis on transdisciplinary collaboration, the new method may facilitate lignin's use as a value-added chemical, rather than a waste product, of biofuel processing (Rahimi et al. 2013).

Understanding and Manipulating Lignin Biosynthesis

GLBRC Plants researchers played a key role in an international research project aimed at re-evaluating the lignin biosynthetic pathway. After researchers at Ghent University in Belgium identified a new gene that produces a previously unknown enzyme (caffeoyl shikimate esterase, or CSE) involved in lignin production, GLBRC researchers used nuclear magnetic resonance technology to analyze Arabidopsis mutants with knocked-out CSE genes. They found that turning off CSE production results in 36% less lignin per gram in Arabidopsis stem tissue, and that the remaining lignin has a significantly altered—and possibly more digestible—composition. Researchers plan to apply these findings to cellulosic bioenergy feedstocks such as poplar to reduce the amount of lignin and increase its digestibility, potentially lowering the cost and energy inputs required to convert cellulosic biomass to ethanol (Vanholme et al. 2013).

Sustainable Catalytic Production of Gamma-Valerolactone

Sustainable Catalytic Production of Gamma-Valerolactone (GVL). GLBRC researchers have used GVL, a recyclable solvent, to extract high yields of sugars from solubilized cellulose. This cellulose conversion approach eliminates the need to separate the final product from the solvent because the GVL product is the solvent. [Image courtesy GLBRC]

Using Green Solvents to Produce Sugars from Biomass

GLBRC Conversion researchers recently succeeded in obtaining valuable linear alpha olefins (LAOs) from an organic solvent called gamma-valerolactone (GVL) in a highly selective catalytic reaction. This process could provide an efficient and renewable option for manufacturers of plastics, detergents, and other traditionally petroleumbased materials (Wang et al. 2013).

Conversion researchers also used a mixture of GVL and water to recover solubilized sugars from biomass. GVL, which is itself obtained from cellulose, is a green solvent and thus can be recycled. High yields of solubilized sugars have been generated from this process, and an initial economic assessment of the technology has indicated that it could produce ethanol at a cost savings of roughly 10% when compared with current state-of-the-art technologies (Luterbacher et al. 2014). With support from the Wisconsin Alumni Research Foundation (WARF), the team will begin scaling up this process later this year. Under the WARF Accelerator Program, GLBRC researchers will construct a high-efficiency biomass reactor that will use GVL to produce concentrated streams of high-value sugars and intact lignin solids. Carbohydrates and lignin from the reactor will be delivered to scientific collaborators, where the fermentation potential of recovered sugars can be tested to create opportunities for integration (Wettstein et al. 2012).

Metabolic Modeling of Microbes

GLBRC Conversion researchers are working to identify microbial strains that can thrive within the highly stressful fermentation environment. Mathematical modeling techniques have provided advances in this area by enabling unique insight into metabolic pathways. In 2012, researchers reported an improved method for predicting metabolic flux changes in key model microbes in response to environmental variation. The method, called RELATCH (RELATive CHange), uses gene expression data to accurately predict genome-scale flux distribution and corresponding enzyme contribution. RELATCH's ability to predict metabolic responses in Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis dramatically outperforms existing methods. These findings demonstrate the applicability of RELATCH for engineering microbial strains with improved biofuel production (Kim and Reed 2012).



Works Cited

Casler, M.D. 2010. "Changes in Mean and Genetic Variance During Two Cycles of Within-Family Selection in Switchgrass," BioEnergy Research 3(1), 47–54.

Gelfand, I., et al. 2013. "Sustainable Bioenergy Production from Marginal Lands in the US Midwest," Nature 493, 514–17.

Jin, M., et al. 2013. "Phenotypic Selection of a Wild Saccharomyces cerevisiae Strain for Simultaneous Saccharification and Co-Fermentation of AFEX-Pretreated Corn Stover," Biotechnology for Biofuels 6, 108.

Kim, J., and J. L. Reed. 2012. "RELATCH: Relative Optimality in Metabolic Networks Explains Robust Metabolic and Regulatory Responses to Perturbations," Genome Biology 13(9), R78.

Rahimi, A., et al. 2013. "Chemoselective Metal-Free Aerobic Alcohol Oxidation in Lignin," Journal of the American Chemical Society 135, 6415–18. DOI: 10.1021/ja401793n.

Takasuka, T. E., et al. 2013. "Aerobic Deconstruction of Cellulosic Biomass by an Insect-Associated Streptomyces," Scientific Reports 3, 1030. DOI: 10.1038/srep01030.

Vanholme, R., et al. 2013. "Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis," Science 341, 1103–06. DOI:10.1126/science.1241602.

Wang, D., et al. 2013. "A Highly Selective Route to Linear Alpha Olefins from Biomass-Derived Lactones and Unsaturated Acids," Chemical Communications 49(63), 7040–42.

Wettstein, S. G., et al. 2012. "Production of Levulinic Acid and Gamma-Valerolactone (GVL) from Cellulose Using GVL as a Solvent in Biphasic Systems," Energy & Environmental Science 5(8), 8199–203.

Zhang, Y., et al. 2011a. "Natural Hybrids and Gene Flow Between Upland and Lowland Switchgrass," Crop Science 51, 2626–41. DOI: 10.2135/cropsci2011.02.0104.

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