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A talented team undertakes a novel strategy to developing alternative fuels.
Oak Ridge National Laboratory microbiologist Tommy Phelps sees the untapped potential of bioenergy in shelves of bottles and beakers containing microscopic organisms that just might hold the elusive bug or enzyme capable of digesting large quantities of plant matter into ethanol. Phelps's current batch of microbes, stockpiled in dozens of bottles of silt, rocks and soils, was collected from Yellowstone National Park, where the hot springs that draw millions of summertime visitors also nurture microscopic life in their boiling waters. These bugs, in turn, beckon microbiologists like Phelps, who seek a solution to transform Earth's abundant cellulosic sources into a modern energy supply. Yellowstone's warm waters offer the promise of microbes that can rapidly and efficiently degrade cellulose—the woody, leafy matter that makes up plants. Scientists hope to tap the power of these microbes for industrial-scale consolidated bioprocessing of plants, including trees and switchgrass, the species central to the BioEnergy Science Center's research efforts. The hunt for this cellulosic "super bug" is part of a suite of efforts under way at the BioEnergy Science Center, headquartered at ORNL. Since being named one of three $135 million Department of Energy bioenergy research centers, researchers at ORNL and its partner institutions have quickly gotten to work. DOE's ambitious goal is to replace by 2030 one-third of the nation's transportation fuel with cellulosebased sources. At these centers, researchers are carrying out the targeted, fundamental science needed to bridge the gap between the potential of cellulose-based fuels and their reality. In addition to ORNL, BioEnergy Science Center partners include the University of Tennessee, the University of Georgia, Georgia Tech, Dartmouth College, the National Renewable Energy Laboratory, ArborGen, Verenium Corp., Mascoma Corp. and the Samuel Roberts Noble Foundation, as well as individual scientists from other institutions. "To develop what we call a consolidated bioprocess for cellulosic ethanol production, we are hoping to reduce the steps required to break down plant matter into sugars and ferment them into ethanol," says Martin Keller, the center's director. "By tapping nature, we hope to replace the current, and expensive, chemical processes with a microbe, enzyme or combination that consolidates multiple steps into one. That is the best path to economical, efficient bioethanol production." Thus, October found Phelps and Keller on the hunt for microbes, a journey that will likely take them to various corners of the globe—through the center's partnership with Verenium—as well as locales closer to home in search of the best plant-processors that nature has to offer. Once collected, these microbes will be exposed to samples of poplar and switchgrass to test their ability to digest the cellulose. Then the most promising bugs will be put through a further gamut of tests to break down and map out their molecular function. Current microbes and enzymes are relatively slow at attacking plant matter's complicated and protective structure. Researchers will determine precisely the genes involved in the interaction of the microbes and enzymes to break apart cellulose. Other genes responsible for producing undesirable products, such as acetic acids, will be knocked out in the hope of, ultimately, developing the perfect ethanol-manufacturing microbe. Particular enzymes will be isolated as well and genetically analyzed, with a focus on determining the ideal formula of enzyme or microbe and enzyme to serve as the vehicle for cellulosic ethanol production. Microbes, however, are just a piece of the puzzle. Other researchers at the Oak Ridge center are going through similar steps to develop plants with qualities most conducive to processing into biofuel. Similar to the microbial work, researchers will analyze thousands of genetically modified switchgrass and poplar tree samples in order to discover and develop the best varieties for ethanol production. As part of the process, the biofeedstock, together with the microbes and the enzymes, will be joined in a complex matrix of analysis and R&D in order to develop the best biofuel recipe. On the biomass formation side, the partners will produce samples of plant material genetically altered to modify their cell walls for optimum breakdown into usable sugars. Such altered species might feature lower amounts of lignin—the substance that holds cellulose fibers together—or a reduction in the crystallinity of the cellulose. ArborGen and ORNL will be primarily responsible for creating and studying various altered trees, while scientists from the University of Tennessee, the University of Georgia and the Noble Foundation will take the lead in switchgrass research. To find rapidly the most important effects and genes, researchers must screen thousands of samples for recalcitrance, says Brian Davison, the BioEnergy Science Center's lead scientist for characterization and modeling. "This task requires us to create a tiered, high-throughput characterization pipeline that reflects the complicated nature of plant recalcitrance, the resistance to breakdown into sugars. The screening must be realistic but practical because we can make improvements in the plants only if we are able to precisely measure their resistance—or lack of resistance—to processing. In other words, you get what you screen for." The samples will be delivered to the National Renewable Energy Laboratory, which will serve as a clearinghouse for cataloging, classification and detailed analysis. Assigned individual bar codes, the samples eventually will make the rounds of various institutions and be divided into separate samples—one ground for air drying, one to image, one shredded for pre-treatment and one frozen in its original state. All samples will then undergo composition analysis, using state-of-the-art techniques such as analytical pyrolysis mass spectrometry or near infrared spectroscopy, to determine how much cellulose versus lignin is present. Next is a pretreatment with dilute acid to replicate the best current processing method developed by the University of California, Riverside. Following the pre-treatment the samples will be chemically assayed again to determine their response to the treatment. Finally, samples will undergo an enzyme digestion assay, which will test for reducing sugar release. This sugar release, along with the preceding measurements, will give researchers a pool of data on recalcitrance and enable subsequent studies to focus on the best samples and the genetic changes that lead to them. Besides more detailed analytical examination with electron microscopy, nuclear magnetic resonance spectroscopy, microCAT scanning and new tools under development, samples that show promise in these tests will be exposed to experimental enzymes and microbes such as those discovered in Yellowstone—which will have undergone a similar rigorous genetic and systems biology analysis—to determine the ones that work best together. Meanwhile, the enormous volume of results from the thousands of biomass, microbe and enzyme samples scooting through the system will be fed into large databases maintained by the various partners and housed primarily at ORNL's Center for Computational Sciences. To make sense of these vast databases, frequent personal communication among the partners is critical. By the end of the 2007, team members had spent more than 40 days in face-to-face meetings to plan a truly cohesive research effort. "We are developing a process that is standardized in order to methodically find and develop the plants, microbes and enzymes that will work the best while ruling out the ineffective ones," Keller says. Summing up the center's opportunity and challenge, Keller adds, "This is the first time anyone has taken this kind of systematic approach to the bioenergy problem." —Larisa Brass Contact: Brian H. Davison
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