"At JBEI, we are inspired by the
vision of moving the nation toward
a sustainable, clean energy future.
The threat of a warming planet and
the need to diversify our energy
sources while supplying transportation
fuels for a growing population
drive our research. JBEI was
designed to be nimble and flexible
enough to focus and refocus our
research quickly and effectively.
Our entrepreneurial culture acts
as a bioscience incubator and has
produced a portfolio of intellectual property in feedstocks,
biomass deconstruction, fuel synthesis, bio-based products,
and enabling technologies that will help fuel the economy
and grow the emerging biofuels industry. Our scientists and
staff envision a future when cellulosic biofuels provide transformative
advantages for our nation."
– Jay Keasling
Jay Keasling is the chief executive officer of JBEI, the associate laboratory director for Biological Sciences at Lawrence Berkeley National Laboratory, and the Hubbard Howe Jr. Distinguished Professor of Biochemical Engineering at the University of California–Berkeley. He is one of the world's foremost authorities on synthetic biology.
The DOE Joint BioEnergy Institute (JBEI) is a partnership led by Lawrence Berkeley National Laboratory (LBNL) that leverages the scientific expertise, resources, and support of four national laboratories and three academic institutions. This multiinstitutional research team is using the latest techniques in molecular biology, chemical and genetic engineering, and computational and robotic technologies to develop advanced biofuels. JBEI consolidates all biomass-to-biofuels research areas within three scientific divisions and one technologies division at its research center in Emeryville, California. Since 2007, JBEI has been addressing key roadblocks to converting lignocellulosic biomass into advanced fuels through an integrated, multidisciplinary approach.
In the Feedstocks Division, researchers are engineering nonfood plants for optimal sugar yields and reduced resistance to breakdown. They have combined two highly targeted bioengineering strategies to redesign the cell walls of plants, resulting in a 20% increase in cell wall sugar content and a 2.5-fold increase in sugar yield. Meanwhile, researchers in the Deconstruction Division have demonstrated that certain ionic liquids—a novel class of biomass solvents—effectively pretreat a wide range of biomass types, facilitating enzymatic conversion to sugars. In a first-of-its-kind effort, they demonstrated a "one-pot," wash-free process that combines biomass breakdown and sugar extraction into a single vessel. This process eliminates the excessive use of water and waste disposal currently associated with washing biomass after ionic liquid pretreatment. Refinement of this one-pot method could significantly simplify and lower the cost of biofuel production processes. In the Fuels Synthesis Division, JBEI's pioneering work in synthetic biology has produced engineered microbes that transform the complex sugars derived from lignocellulosic biomass into biofuels that can directly replace petroleum-based gasoline, diesel, and jet fuel. These advanced biofuels do not require modification of today's engines or fuel infrastructures and can be incorporated with no loss of performance. JBEI has produced a portfolio of intellectual property in feedstocks, biomass deconstruction, fuels synthesis, and enabling technologies to help advance the emerging biofuels industry.
JBEI brings the sunlight-to-biofuels pipeline under one roof in four interdependent research divisions that focus on (1) designing and developing new bioenergy crops, (2) enhancing biomass deconstruction, (3) developing routes to new biofuels through synthetic biology, and (4) creating technologies that advance biofuels research.
Researchers in JBEI's Feedstocks Division are improving the understanding of genes and enzymes involved in the synthesis and modification of plant cell walls using well-characterized genomes and genetic-engineering tools for rice and Arabidopsis (a small flowering plant related to mustard). These two model systems are ideal for research because their development from seed to mature plant takes only weeks or months, rather than the year or more required for crops such as switchgrass and poplar. Genetic insights from rice (a model for grasses and other monocots) and Arabidopsis (a model for trees and other dicots) will accelerate the breeding of new energy feedstocks (see figure, Developing Better Plants for Biofuels, below). This knowledge is used in developing specialized fuel crops that are optimized for deconstruction into sugars and fermentation into biofuels and can thrive with little fertilization or irrigation on land not suitable for growing food crops.
In addition, scientists are investigating metabolic pathways involved in the biosynthesis of lignin, a tough structural material that shields a plant's energy-rich sugars from enzymatic attack. This unique basic research program could help transform lignin into a valuable source of chemicals and polymers while improving the economics of converting cellulosic biomass into fuels.
Developing Better Plants for Biofuels. April Liwanag, a research assistant in JBEI's Feedstocks Division, checks on seedlings of Arabidopsis, one of two model plants researchers are studying in efforts to design improved bioenergy crops. [Image courtesy JBEI]
Unlike the simple starch-based sugars in corn and other grains, the complex polysaccharide sugars in nonedible plant cell walls are locked within lignin. The process of liberating fermentable sugars that can then be converted into fuels is called "deconstruction" and normally requires two steps: pretreatment and enzymatic hydrolysis. The Deconstruction Division is using ionic liquids (molten salts that are liquid at room temperature) as a novel means of pretreating biomass. Free of the inhibitors associated with other pretreatment methods, ionic liquids help liberate high yields of fermentable sugars that can be converted to biofuels and biochemicals. Moreover, ionic liquid pretreatment is the only known technology that can efficiently process a wide range of single and mixed feedstocks, including agricultural and forest residues, grasses, and woody perennials. Researchers are investigating the effects of ionic liquids on cellulosic biomass and the recovery of sugars and lignin through selected liquid-liquid extraction and filtration. Using advanced imaging and spectroscopy, coupled with computer simulations, scientists are obtaining a detailed, cellular-level understanding of the ionic liquid pretreatment process and are using this information to develop scalable and cost-competitive engineering solutions.
Another key aim of the Deconstruction Division is developing cost-effective enzyme mixtures that will tolerate potential biorefinery conditions, such as extremes of temperature and pH or the presence of ionic liquids. JBEI scientists also are exploring a range of ecosystems, from rainforest floors to composts, to discover and isolate new microbial enzymes that can efficiently degrade cell wall polysaccharides and lignin. Studies of fungal biotechnology are under way to generate a genetic toolbox for more efficient protein production in fungi to expedite the discovery of high-performance industrialstrength enzyme mixtures that operate effectively at high biomass loading.
JBEI researchers are applying synthetic biology techniques and mathematical models of metabolism and gene regulation to engineer microorganisms to efficiently convert sugars released from deconstructed biomass into the energy-rich molecules of advanced biofuels such as alkanes. Scientists in the Fuels Synthesis Division are engineering new strains of Escherichia coli, the gut bacterium, and yeast, the common single-cell organism used in baking bread and making beer, to more quickly and efficiently ferment the sugars derived from cellulosic biomass into biofuels. Their goal is to produce advanced biofuels that (1) yield as much energy per volume as petroleum-based fuels, (2) can be shipped through existing fuel pipelines, and (3) can burn in existing engines. Biologically produced alkanes and other oil-like hydrocarbons could replace gasoline, diesel, and jet fuel on a gallon-for-gallon basis. Work also is being done to develop experimental wetware, software, and laboratory automation devices that facilitate, accelerate, and standardize the engineering of microbes that produce fuels from cellulosic biomass.
Improving Access to Energy-Rich Sugars. Ning Sun is part of a team of researchers in JBEI's Deconstruction Division exploring the use of ionic liquids to pretreat biomass. [Image courtesy JBEI]
JBEI scientists are devising new, broadly applicable technologies to accelerate research that will lead to renewable biofuels. Among these are techniques for characterizing genes and proteins in both natural and engineered plants and microorganisms. Innovations include chip-based systems for identifying new enzymes with cellulose- and lignin-degrading activities. Researchers also have developed high-throughput methods using microfluidics and array platforms that can screen hundreds of enzymatic reactions simultaneously to help identify the best enzymes for biomass deconstruction. Highresolution imaging visualizes in greater detail plant cell walls and their components and also helps characterize the effects of pretreatment protocols on plant biomass. These and other enabling technologies are generating large volumes of data collected in a centralized database for computational analysis.
Many of JBEI's numerous successes during its first 6 years of operation stem from pioneering work in synthetic biology. Sometimes considered a new field, the concept of synthetic biology can be traced back to when humans first began trying to improve the usefulness, efficiency, or performance of things in the natural world around them. The goal has always been to change existing materials and resources or create new ones that do what raw resources cannot. Today, synthetic biologists are designing and building biological systems for specific purposes, including biofuels. Since 1992, Jay Keasling, JBEI's chief executive officer, has been redesigning microbes to operate as miniature chemical reactors that transform sugars into valuable products. "It's a lot like brewing beer from yeast and hops. But rather than alcohol being the end product, you program your microbe to produce fuels," Keasling says. "Our goal at JBEI has been to put as much chemistry as we can into microbes. We graft genes from plants into the microbes. Once inside, the genes produce enzymes that do the chemistry to transform sugars into fuel," he says. "Enzymes can do in one step what might take many steps using synthetic organic chemistry." Prominent achievements include engineering the first strains of Escherichia coli that can digest switchgrass and synthesize its sugars into gasoline, diesel, or jet fuel without any help from enzyme additives. Drawing on advances in molecular, cell, and systems biology, the growing synthetic biology toolset provides critical new insights into the natural world. Synthetic biology techniques also have been used to engineer plants that produce more cellulose; less hemicellulose; and lessrecalcitrant lignin, a trait enabling easier extraction of sugar-containing cellulose and hemicellulose. Together, these advances will increase sugar yields from energy crops, thereby reducing the cost of fuels derived from them. The promise of synthetic biology is the ability to turn any plant, including nonfood sources such as forest debris, weeds, even paper waste, into energy.
Accelerating the transfer of JBEI inventions to private industry is a critical aspect of the institute's mission. An innovative culture has resulted in collaborations with industry and a wealth of intellectual property available for licensing. Companies sponsoring research have access to world-class scientific expertise, capabilities, and infrastructure to speed time to market. JBEI researchers also stay current on industry challenges and opportunities by interacting with members of the Industry Advisory Committee who represent the entire spectrum of the biofuels industry including energy, agribusiness, and biotechnology. JBEI research is informed by real-world needs and standards through ongoing talks with representatives from a broad range of companies. To facilitate technology transfer, JBEI's director of commercialization is authorized to implement licensing agreements for all of JBEI's institutional partners. As of spring 2013, JBEI has partnered with 29 companies under a variety of arrangements and has spun off three startup companies. In addition, the rate of JBEI invention disclosures is trending at 2.5 times the rate of the top four U.S. universities, while the rate of patent licenses is two times greater (Association of University Technology Managers FY 2011–12 survey of academic institutions, www.autm.net).
To expedite commercialization of its research breakthroughs, JBEI conducts feasibility and scale-up tests using the facilities at LBNL's Advanced Biofuels Process Demonstration Unit (ABPDU). Located in Emeryville, ABPDU provides industry-scale test beds for discoveries made in the laboratory, including JBEI's work on ionic liquid pretreatment and microbial synthesis of bisabolene, a terpene-based precursor to an advanced biofuel that potentially could replace diesel.
In a study assessing the optimization and scaling potential of ionic liquid pretreatment, JBEI and ABPDU scientists and engineers determined that this technology can be effectively scaled to larger biorefinery operations (Chenlin et al. 2013). Using the study as a baseline, researchers will develop and apply improved biomass deconstruction processes to diverse feedstocks as a means to realize a commercially viable pretreatment method based on ionic liquids. In addition, unit operations and process parameters obtained in the study will be essential for analyzing the technoeconomic aspects of this technology. The JBEI-ABPDU collaboration is the first of its kind in the research community, successfully identifying key opportunities and challenges associated with ionic liquid pretreatment and subsequent enzymatic saccharification beyond the bench scale.
JBEI also is working with ABPDU to scale up microbial production of bisabolene, a member of the terpene class of chemicals found in plants and used in fragrances and flavorings. When hydrogenated, bisabolene becomes bisabolane, a compound with fuel properties comparable to commercial diesel and also considered a promising jet fuel. Using the tools of synthetic biology, JBEI scientists engineered microbes that produce bisabolene from a simple carbon source. To increase production, JBEI collaborates with ABPDU, which has a sequential scale-up capacity from 1 L to 300 L. The team is working with an industrially favored engineered yeast strain, aiming to generate 1 gallon of the biofuel for jet fuel specification.
JBEI is working to keep our nation at the vanguard of scientific discovery by developing future generations of scientists. Preparing a multidisciplinary workforce of researchers is key to growing the cellulosic biofuels industry.
Year-round, JBEI scientists and staff provide educational opportunities (e.g., site tours, workshops, presentations, and educational programs) for high school to graduate students. Opportunities to explore careers in science and engineering are offered through direct mentoring and internships for undergraduate and graduate students. Each summer, JBEI partners with the Synthetic Biology Engineering Research Center (SynBERC) to manage an intensive internship program for high-potential, low-income high school students from communities underrepresented in science. Guided by JBEI scientists, students in the Introductory College-Level Experience in Microbiology (iCLEM) program participate in hands-on biofuels research. During the program's first 5 years, nearly 100% of iCLEM interns have enrolled in college, and 63% have elected to major in science or engineering.
Through ongoing, in-house professional development, JBEI offers weekly seminars and workshops to enhance research performance, collaboration, and results. Industry leaders are invited to give entrepreneurial training as part of guest lectures focused on enabling young scientists and engineers to develop biofuels-related business plans and advance their careers.
Engineering Enhanced Plants. JBEI researchers genetically engineered Arabidopsis plants (#89) that yielded as much biomass as wild types (WT) but with increased polysaccharide deposition in the fibers of their cell walls. [Images courtesy JBEI]
Lignocellulosic biomass is composed mostly of plant secondary cell walls that contain, among other polymers, complex chains of sugars called polysaccharides that hold great promise as biofuel sources. These sugars, however, are embedded in lignin, a tough material that helps reinforce cell wall structure and maintain its integrity. Because lignin is primarily responsible for the resistance of biomass to enzymatic hydrolysis or breakdown, considerable research has been directed at decreasing the amount of lignin in the cell wall. Previous efforts have demonstrated that large reductions in lignin content cause growth defects and often correlate with vessel collapse, adversely affecting water and nutrient transport carried out by the plant vascular system.
JBEI researchers have developed a new approach to decrease lignin content while preventing vessel collapse and have introduced a novel strategy to boost transcription factor expression in specific tissues. Synthetic biology tools were used to rewire the secondary cell network in Arabidopsis by changing promoter-coding sequence associations. The result was a reduction in lignin and an increase in polysaccharide depositions in the plant's fiber cells. The promoter of a key lignin biosynthesis gene, C4H, was replaced by the vessel-specific promoter of transcription factor VND6. This replacement rewired lignin biosynthesis specifically for vessel formation while disconnecting C4H expression from the fiber regulatory network. Secondly, the promoter of the IRX8 gene, encoding secondary cell wall glycosyltransferase, was used to express a new copy of the fiber transcription factor NST1. When the IRX8 promoter is induced by NST1, it also creates an artificial positive feedback loop (APFL). The combination of strategies—lignin rewiring with APFL insertion—enhances polysaccharide deposition in stems with low lignin content, resulting in higher sugar yields after enzymatic hydrolysis. If commercialized, such plants would increase biofuel yield per acre and enhance profitability (Yang et al. 2013).
One-Pot Process. The right combination of enzyme cocktail and ionic liquid pretreatment can be used to extract fermentable sugars from switchgrass in a single, wash-free step. [Image courtesy Lawrence Berkeley National Laboratory]
Biomass pretreatment using certain ionic liquids, such as 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), can be highly effective at reducing the recalcitrance of lignocellulosic biomass to enzymatic degradation. However, current commercial enzyme cocktails—derived from filamentous fungi and developed for dilute acid pretreatment—are inhibited by the most effective ionic liquids, whose removal from pretreated biomass requires excessive amounts of water for efficient enzyme performance. The costs associated with ionic liquid recycling and waste disposal pose significant economic and engineering challenges for the commercial scale-up of pretreatment technologies based on ionic liquids.
In a first-of-its-kind effort, JBEI researchers have demonstrated a wash-free process that combines, in a single vessel, ionic liquid pretreatment and saccharification (the breakdown of plant polysaccharides into simple sugars). After treating switchgrass with [C2mim] [OAc] and diluting with water to a final ionic liquid concentration of 10% to 20%, the pretreatment slurry was directly hydrolyzed using a thermostable, ionic liquid–tolerant enzyme cocktail previously developed at JBEI. Within three days, this "one-pot" process liberated 81.2% of the available glucose and 87.4% xylose (monomers and oligomers) at 70оC, with an enzyme loading of 5.75 mg/g of biomass at 10% [C2mim][OAc]. Glucose and xylose were selectively separated by liquid-liquid extraction with over 90% efficiency, thus eliminating extensive water washing as a unit operation. This process could drastically simplify the downstream recovery of sugar and lignin and the recycling of ionic liquids, paving the way for an affordable and scalable approach to producing fermentable sugars from lignocellulosic biomass using ionic liquids (Park et al. 2012).
Identifying New Biofuel Candidates. JBEI researchers Harry Beller (foreground) and Ee-Been Goh have engineered Escherichia coli bacteria to synthesize large quantities of methyl ketone compounds that could be used as biofuels. [Image courtesy Lawrence Berkeley National Laboratory]
JBEI synthetic biologists have engineered Escherichia coli bacteria to generate significant quantities of methyl ketone compounds from glucose. These compounds have high cetane numbers (a diesel fuel rating comparable to the octane number for gasoline), making them strong candidates to replace petroleum-based diesel. Researchers successfully increased the methyl ketone titer production of E. coli more than 4,000-fold with a relatively small number of genetic modifications. These findings add to the list of naturally occurring chemical compounds that could serve as biofuels, providing more flexibility and options for the biofuels industry (Goh et al. 2012).
Synthetic biology methods also were used by researchers in the Fuels Synthesis Division to devise a new technique—called a dynamic sensor-regulator system (DSRS)—that can detect metabolic changes in microbes during the production of fatty acid–based fuels or chemicals. Because DSRS also enables researchers to control the expression of genes affecting this production, the team was able to demonstrate a threefold increase in microbial synthesis of biodiesel from glucose. DSRS is the first example of a synthetic system that can dynamically regulate a metabolic pathway for improving production of fatty acid–based fuels and chemicals while microbes are in a bioreactor (Zhang, Carothers, and Keasling 2012).
High-Speed, Large-Scale Enzyme Screening. Codeveloped by JBEI researchers, high-throughput nanostructure-initiator mass spectrometry (HT-NIMS) can be used to quickly and precisely determine the molecular composition of thousands of samples arrayed on a small slide of silicon. JBEI researchers are using the technology to screen for enzymes useful in biomass deconstruction. [Image courtesy Lawrence Berkeley National Laboratory]
High-throughput nanostructure-initiator mass spectrometry (HT-NIMS), codeveloped at JBEI, is a high-speed chemicalscreening technology that researchers can use to discover the function of large numbers of biologically active molecules. With speeds 100 times faster than conventional probes, HT-NIMS can rapidly screen tens of thousands of enzymatic biomass deconstruction reactions that could be used to turn grass into biofuels.
A workhorse of biotechnology, mass spectrometry (MS) offers unparalleled accuracy, but its potential as a screening tool has been limited by its slow throughput. HT-NIMS dramatically speeds up the process. Tiny sample volumes are deposited in rows and columns on a slide of silicon, creating a microarray of as many as 10,000 discrete sites. Each site is probed with a laser and analyzed in a split second. In the time it takes conventional MS to characterize one sample, HT-NIMS can cost-effectively profile hundreds. JBEI is using the technique, which was recognized with a 2013 R&D 100 Award, to screen for enzymes that can be used to modify lignocellulose for producing advanced biofuels that could replace gasoline on a gallon-for-gallon basis (Greving et al. 2012; Reindl et al. 2012).
Accelerating Commercialization of Research Advances. JBEI's wiki-based technoeconomic model simulates critical factors in the biorefinery process, enabling scientists to evaluate the real-world potential of research developments and focus on the most promising strategies. [Image courtesy JBEI]
To evaluate the potential economic impact of JBEI technological advances, researchers have developed a technoeconomic model as a principal tool for measuring progress. This publicly available model (econ.jbei.org) can be used to simulate the performance and cost-competitiveness of engineered feedstocks, new biomass pretreatments, enzyme mixtures, enzyme loading, and type of biofuel produced. Another model representing the corn stover–to–ethanol process (i.e., dilute acid pretreatment, saccharification, and yeast fermentation) serves as the technical benchmark against which JBEI research developments can be measured. The primary metric for comparing the different biofuel technologies is the minimum ethanol selling price (MESP)—or the price at which a biorefinery can be economically viable. JBEI research advances are incorporated into the technoeconomic model, which calculates the resultant minimum biofuel selling price. This price is then compared with the benchmark MESP for the corn stover process, thereby providing an important measure of progress as it relates to potential commercialization of the technology evaluated.
Greving, M., et al. 2012. "Acoustic Deposition with NIMS as a High-Throughput Enzyme Activity Assay," Analytical and Bioanalytical Chemistry 403(3), 707–11.
Park, J. I., et al. 2012. "A Thermophilic Ionic Liquid–Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels," PLoS ONE 7(5), e37010.
Reindl, W., et al. 2012. "Nanostructure-Initiator Mass Spectrometry (NIMS) for the Analysis of Enzyme Activities," Current Protocols in Chemical Biology. DOI: 10.1002/9780470559277.ch110221.
Yang, F., et al. 2013. "Engineering Secondary Cell Wall Deposition in Plants," Plant Biotechnology Journal 11, 325–35. DOI: 10.1111/pbi.12016.
Zhang, F., J. M. Carothers, and J. D. Keasling. 2012. "Design of a Dynamic Sensor-Regulator System for Production of Chemicals and Fuels Derived from Fatty Acids," Nature Biotechnology 30(4), 354–59. DOI: 10.1038/nbt.2149.
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