Genomic Science Program

Cellulosic Ethanol

Cellulose Degradation and Conversion

Understanding the conversion of biomass to ethanol begins with understanding the structural and chemical complexity of the three primary polymers that make up plant cell walls: Cellulose, hemicellulose, and lignin (see Cellulose Structure and Hydrolysis Challenges). Depending on plant species and cell type, the dry weight of a cell wall typically consists of about 35 to 50% cellulose, 20 to 35% hemicellulose, and 10 to 25% lignin (Saha 2004). Cellulose is the most abundant biomaterial on earth. Each cellulose molecule is a linear polymer of glucose residues. Depending on the degree of hydrogen bonding within and between cellulose molecules, this polysaccharide is found in crystalline or paracrystalline (amorphous) forms. Cellulose exists within a matrix of other polymers, primarily hemicellulose and lignin. Hemicellulose is a branched sugar polymer composed of mostly pentoses (five-carbon sugars) and some hexoses (six-carbon sugars). Lignin is a complex, highly cross-linked aromatic polymer that is covalently linked to hemicellulose, thus stabilizing the mature cell wall. These polymers provide plant cell walls with strength and resistance to degradation, which also makes these materials a challenge to use as substrates for biofuel production.

Enzymes such as cellulases, hemicellulases, and other glycosyl hydrolases synthesized by fungi and bacteria work together in a synergistic fashion to degrade the structural polysaccharides in biomass. These enzyme systems, however, are as complex as the plant cell-wall substrates they attack. For example, commercial cellulase preparations are mixtures of several types of glycosyl hydrolases, each with distinctly different functions (exocellulases, endocellulases, exoxylanases, endoxylanases, cellobiases, and many others). Optimization of these enzymes will require a more detailed understanding of their regulation and activity as a tightly controlled, highly organized system.

The biochemical conversion of biomass to ethanol currently involves three basic steps: (1) thermochemical treatments of raw lignocellulosic biomass to make the complex polymers more accessible to enzymatic breakdown; (2) production and application of special enzyme preparations (cellulases and hemicellulases) that hydrolyze plant cell-wall polysaccharides to a mixture of simple sugars; and (3) fermentation, mediated by bacteria or yeast, to convert these sugars to ethanol. A more complete understanding of enzymes and microbes involved in biomass conversion to ethanol is needed to overcome many current inefficiencies in the production process.

Bioethanol Research Targets for Genomic Science

Improving Cellulase Systems. Genomic Science will accelerate the development of optimal cellulase systems by providing resources for screening thousands of natural and modified enzyme variants, enabling the high-throughput production and functional analysis of these enzymes, elucidating regulatory controls and essential molecular interactions, and developing models for analyzing the structure and activity of natural and engineered enzyme systems.

Enabling the Development of Integrated Bioprocessing. A long-term target for Genomic Science research is integrated bioprocessing, the conversion of biomass to ethanol in a single step. Accomplishing this requires the development of a genetically modified, multifunctional organism or a stable mixed culture capable of carrying out all biologically mediated transformations needed for the complete conversion of biomass to ethanol.

Gaps in Scientific Understanding

Without improving our understanding of microbial processes essential to bioethanol production, developing and improving technologies based on this understanding will be difficult. Biotechnology innovation requires basic research that explores a greater variety of enzymes and microorganisms, analyzes enzymes as systems, and determines how certain factors influence biomass degradation or ethanol production. Several fundamental scientific questions in need of further investigation include:

• What is the extent of natural diversity among biomass-degrading and ethanologenic organisms? Over the last 30 years, most research devoted to ethanol production from cellulose has focused on fungal systems (primarily Trichoderma reesei) for the breakdown of cellulose into sugars coupled with the sugar-fermentation processes of yeast (Saccharomyces cerevisiae) (Demain et al. 2005). A deeper understanding of a greater variety of cellulolytic and ethanologenic systems is needed. Bacterial species in diverse physiological groups (e.g., bacteria with various tolerance levels for oxygen, temperature, and salt concentrations) are known to hydrolyze cellulose; thus a wide range of natural habitats could be explored for novel cellulotyic activities in bacteria.
• How do soluble enzymes act on an insoluble crystalline substrate? The hydrolysis of crystalline cellulose is the rate-limiting step in biomass conversion to ethanol because aqueous solutions of enzymes have difficulty acting on this insoluble, highly ordered structure. Cellulose molecules in their crystalline form are packed so tightly that enzymes and even small molecules such as water are unable to permeate the structure.
• How do different biomass-degrading enzymes work together as a synergistic system? Cellulases and hemicellulases are secreted from cells as free enzymes or as large, extracellular complexes known as cellulosomes. The collective activity of these enzyme systems is much more efficient than the individual activity of any isolated enzyme; therefore, to truly understand how these enzymes function, they must be studied as systems rather than individually or a few at a time. In addition, these systems eventually must be analyzed under laboratory conditions more representative of real-world environments. For example, laboratories often use purified cellulose as the substrate for enzyme analysis rather than more heterogeneous, natural lignocellulosic materials, and this can provide erroneous conclusions about natural enzyme activity.
• Why are ethanologenic organisms less efficient at using certain sugar substrates? A varied mix of hexoses (e.g., glucose, mannose), pentoses (e.g., xylose, arabinose), and oligosaccharides is released from the hydrolysis of lignocellulosic materials, and no microorganism is capable of fermenting all these sugars. The most widely studied ethanologenic microbes (e.g., yeast) prefer to use glucose as a substrate. Even when yeast cells are modified genetically to use xylose, they ferment all glucose before switching to the much slower xylose fermentation. Conversion rates can vary greatly depending on such factors as the type of sugar substrate being fermented, environmental conditions (e.g., pH, temperature), and the concentrations of certain products from other metabolic pathways.
• How effective are sugar transporters at translocating different sugars across the cell membrane? Sugar transporters are membrane-bound proteins that take up sugars from the environment and deliver them to the metabolic pathways inside cells. The inefficient transport of different sugar substrates by microbes can result in low product yield and is a major obstacle to the efficient conversion of biomass to ethanol. Our limited understanding of sugar transporters is due to a lack of adequate techniques for producing membrane proteins and studying their structure and function. Questions in need of investigation include: Can a glucose transporter transport other sugars, and, if so, how efficiently? Are some transporters better than others? Can transporters be modified for improved function?
• Why do different enzymatic and microbial processes operate optimally at different temperatures? Cellulases operate optimally at temperatures (>40°C) higher than those tolerated by ethanologenic organisms, so these two processes currently cannot be consolidated into a single process step. Thermophily (tolerance of high temperatures) improves the robustness of enzymes or microbes needed for industrial-scale processes and reduces the likelihood of culture contamination. The basis by which enzymes, pathways, and entire microbes are made thermophilic is understood poorly, and methods for inserting cellulolytic or fermentative pathways into thermophilic organisms are not well developed.
• What are the requirements for producing and maintaining stable mixed cultures? At a minimum, cultures used in bioethanol-production systems will need to be resistant or stable despite contamination by “outside” microbes or other potentially toxic materials or life forms. We currently do not understand in sufficient detail the dynamics of microbial consortia that carry out stable mixed processes such as aerobic and anaerobic digestion. Without this understanding, we will not be able to “design” or “engineer” such systems.
• How can we improve systems for genetically engineering microorganisms involved in bioethanol production? While many studies have expressed genes from cellulolytic organisms in Escherichia coli or other mesophilic organisms, systems for expressing foreign genes in cellulolytic or thermophilic organisms are in need of further development. Our current limited understanding of microbial regulation prevents the successful engineering of a microbe capable of versatile expression of lignocellulolytic enzymes, utilization of multiple sugars, and glycolysis.

Scientific and Technological Capabilities Required to Achieve Goals

Improving current understanding of bioethanol production will require a variety of new capabilities including techniques for surveying enzyme diversity; visualizing enzyme systems; efficiently producing enzyme systems and membrane proteins; cultivating microbial consortia; integrating transcriptomics, proteomics, and metabolomics; and genetically engineering microorganisms for integrated bioprocessing. Specific needs include the following:

• Ecogenomic approaches to explore the natural diversity of cellulases. High-throughput sequencing and computational analysis of DNA from environments in which cellulose is widely available will lead to the discovery of genes for novel cellulase systems that could be used as templates for protein production.

• Techniques to visualize cellulase systems in motion. Advanced imaging techniques will provide new insights into how cellulases interact with crystalline cellulose and overcome current barriers to efficient cellulose hydrolysis (e.g., substrate accessibility, product or substrate inhibition, low product yield). Structural information and imaging from X-ray, nuclear magnetic resonance spectroscopy, scanning transmission electron microscopy, and other techniques will be needed to identify additional interactions between cellulases and other molecules needed for efficient function.
• Large-scale production of cellulase enzyme systems, sugar transporters, and other proteins. This will require improved methods for protein production and characterization. Currently, synthesis of sugar transporters and other membrane proteins is difficult, so analyzing the structure and activity of these proteins is challenging, if not impossible. High-throughput techniques and expression systems for efficiently producing membrane proteins, sets of different enzymes that work together, and enzyme complexes such as cellulosomes are in need of development. Access to validated expression systems for microorganisms with mission-relevant capabilities, including thermophilic, cellulolytic, and ethanologenic organisms, would help researchers spend less time on developing expression methods and more time on characterizing and improving proteins.
• Methods to grow stable mixed cultures. Improved experimental and modeling tools are needed to develop methods for producing a mixed microbial culture. The goal is to enable each population carrying out one part of the overall ethanol production process to perform stably.
• Methods to integrate transcriptomic, proteomic, and metabolomic information. Techniques that integrate information gathered from these global molecular measurements are essential to determining which genes are expressed and functionally active during cellulose utilization or ethanol fermentation and which metabolites influence the activity of enzymes involved in these pathways. As an insoluble substrate, cellulose cannot enter cells and induce the expression of genes involved in cellulose hydrolysis. Metabolic profiling could be used to identify which substrates or metabolites at what quantities activate or repress expression of key cellulolytic genes. In addition to illuminating regulatory strategies for cellulases and other coexpressed enzymes such as ligninases, these integrated omic approaches could be used to build regulatory and metabolic maps to guide genetic engineering. For example, these maps could be used to identify the best potential gene knockouts that redirect carbon flux from a particular sugar substrate toward ethanol fermentation and bypass competing pathways that produce other organic end products.
• Methods to genetically engineer organisms for integrated bioprocessing. Clostridium thermocellum is an anaerobic bacterium capable of both hydrolyzing cellulose and fermenting sugars to ethanol, but its yields are poor and conversion is slow. Improved methods for genetically modifying this and other cellulolytic microbes are needed. In one approach to developing an organism for integrated bioprocessing, a microbe naturally capable of hydrolyzing cellulose, such as C. thermocellum, is engineered to provide high product (ethanol) yields. In another approach, noncellulolytic microorganisms known to have high yields of ethanol are engineered to express cassettes of genes encoding cellulase enzyme systems. In either case, to achieve this ambitious goal of developing an organism capable of integrated bioprocessing, the current research paradigm must be altered to focus on understanding how microbial systems function and how their interacting pathways influence one another rather than focusing on only a few genes or enzymes.

Text adapted from Genomics:GTL Roadmap: Systems Biology for Energy and Environment, U.S. Department of Energy Office of Science, August 2005. DOE/SC-0090.